INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Quinnipiac College IMMUNOPEROXIDASE STAINING FOR CYTOKERATIN USING THE MICROWAVE OVEN By Susan M. Mitchell B .A . Assumption College, 1984 A THESIS Presented to the School of Allied Health and Natural Sciences and Quinnipiac College in partial fufillment of the requirements for the degree of Master of Health Science May 1986 S' m Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 1414083 __ ® UMI UMI Microform 1414083 Copyright 2003 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT IMMUNOPEROXIDASE STAINING FOR CY TOKERATIN USING THE MICROWAVE OVEN Susan M. Mitchell Master of Health Science School of A l l i e d Health and Natural Science Q u i n nip iac College May 19 8 6 Cytokeratin is a structural protein found primarily in epithelial cells. Its presence is used to identify the origin of certain tumors. Cytokeratin can be d ete c t e d by the immunoperoxidase technique, (PAP) a valuable but time consuming procedure. A microwav£ oven was utilized in this proje ct in an attempt to decrease immunoperoxidase staining time. Skin of human thigh was stained with commercially pre pared PAP reagents for cytokeratin incorporating brief m icrowave exposures at certain steps. The tolerance of P A P antibody and enzyme reagents to microwave irradiation was evaluated. Individual brief m icrowave treatments of sections covered with PAP reagents was not harmful to the tissue or reagents. A 20 second microwave exposure, prior to routine incubation of the primary antibody, a n t i - c y t o k e r a t i n , was found to significantly enhance the stain intensity. However, Q U IN N IPIA C COLLEGE LIBRARY HAMDEN, CT. 06518 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 164 0 accumulations of repeated microwave treatments appeared to destroy the tissue or the reagents, or both, as reflected by considerable non-specific background staining. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IMMUNOPEROXIDASE STAINING FOR CYTOKERATIN USING THE MICROWAVE OVEN This thesis is approved as a credible and independent invest igation by a candidate for the degree of Master of Health Sciences, and is acceptable as meeting the thesis requirements for this degree, but without implying that the conclusions reached by the candidate are necessarily the conclusions of the major department. Qjinnipiac College Thesis Advisor Irwin Beitch, Ph.D. Professor of Biology Department of Medical Laboratory Sciences Quinnipiac College Clinical Thesis Advisor Leo J. Kelly, M.H.S. Clinical Coordinator - Pathologists' Assistants Training Program, Quinnipiac College and West Haven Veterans Administration Medical Center Medical Director R jtvscb. ___________________ Pathologists' Assistants Training Program Rosa E. Enriquez, M.D. Director of Pathologists' Assistants Training Program West Haven Veterans Administration Medical Center Assistant Professor of Pathology, Yale University Quinnipiac College Edward Kavanagh, Ph.D. Assistant Professor of Biology ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I wish to express my appreciation to the following individuals for their help and assistance in the completion of this thesis project. To Rosa Enriquez, M . D . , whose guidance and professional knowledge played a major role in the initiation and form ulation of this project. To Leo Kelly, patience, M . H . S . , who generously offered his time, and insight to me throughout this project, as well as over the past two years. To Irwin Beitch, P h . D . , for sharing his kn owledge and experience to help d evelop a scientifically sound research project and a well wr it t e n paper. To Kent Marshall, Ph.D. and Edward K a v a n a g h , P h . D . , for their technical guidance and knowledge that were essential for proper completion of this project. To my parents, continuous interest, my endeavors, Mr. and Mrs. Carl M i t c h e l l , for their support, and encouragement through all especially over the past two years. i ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Page I N TRO DUC TI ON................................................... 1 Statement of the P r o j e c t ................................1 Literature R e v i e w ........................................ 2 Microwave Energy and H e a t i n g ..................... 2 The Effect of Microwave Radiation on Biological M o l e c u l e s ..................... 3 Laboratory Use of a Microwave O v e n .............. 6 Immunocytochemis t r y ................................7 C y t o k e r a t i n ........................................ 11 The S k i n ........................................... 13 Immunoperoxidase S t a i n i n g ........................15 Peroxidase-Antiperoxidase Staining for C y t o k e r a t i n ............................. 17 R a ti on ale ................................................ 21 MATERIALS AND M E T H O D S ........................................ 24 R E S U L T S ........................................................ 31 Effects of Microwave Treatment on Individual PAP Serum R e a g e n t s ......................... 31 Physical T o l era nce of Tissue Sections and PAP Serum Reagents to Microwave E x p o s u r e ...... 31 Individual Micr owa ve Irradiation of PAP Serum Reagents With Subsequent Routine I n c u b a t i o n s ...............................31 Microwave Irradiation of PAP Serum Reagents Without Routine I n c u b a tio ns ..................... 33 Effects of Microwave Treatment on Combinations of PAP Serum Reagent S t e p s ............................ 33 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS (continued) Page Combinations of Microwave Treatments Which Were Shown to Give Positive Results I n d i v i d u a l l y........................................33 Microwave Treatment of Ail PAP Serum Reagents Except the Primary A n t i b o d y ............... 39 Microwave Staining of Five Surgical Specimens Containing C yt oke rat in ................. 39 D I S C U S S I O N ......................................................46 Data A n a l y s i s ............................................ 46 Overall C o n c l u s i o n s ..................................... 56 RE F E R E N C E S ...................................................... 58 APPENDIX 1 ......................................................61 APPENDIX I I .....................................................62 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. L IS T OF T A B L E S Page Table 1. A comparison of routine reaction times and microwave reaction times for specific steps in some staining p r o c e d u r e s ................. 8 Table 2. Functions and properties of the five classes of immunoglobulin p r o t e i n s ......... 12 Table 3. Individual microwave irradiation of PAP serum reagents with subsequent routine i n c u b a t i o n s ................................ 3 2 Table 4. Microwave irradiation of PAP serum reagents without subsequent routine in cub at ion s.......... 35 Table 5. Further microwave testing of anti-cytokeratin and peroxidase-antiperoxidase r e a g e n t s .......... 36 Table 6. Methods used to reduce reaction time of anti-cytokeratin r e a g e n t ................. 37 Table 7. Combined microwave treat me nt PAP p r o c e d u r e .....3 8 Table 8. Results obtained using combined microwave treatment for all PAP serum r eag en ts ............ 40 Table 9. Combined microwave tr eatment PAP procedure for all steps except the primary antibody s t e p ....................................... 41 9 Table 10. Results obtained using combined microwave treatment for all PAP reagents except a n t i - c y t o k e r a t i n ........................... 42 Table 11. Comparison of routine and microwave staining of specimens known to contain c y t o k e r a t i n ................................ 45 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Page s p e c t r u m ...................... 4 Figure 1. The electromagnetic Figure 2. Schematic drawing of an antibody m o l e c u l e Figure 3. Cell layers of s k i n ............................... 16 Figure 4. Schematic diagram of a peroxiaaseantiperoxidase m o l e c u l e .......................... 20 Figure 5. Schematic diagram of the peroxidaseantiperoxidase staining p r oce du re .............. 22 vi i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10 L IS T OF PLATES Page Plate 1. Human skin from thigh stained by routine PAP procedure for c y t o k e r a t i n ....................25 Plate 2. Illustration of grading scale used to evaluate specific c y t o k er at in stain reaction in a PAP p r o c e d u r e ...................... 26 Plate 3. Illustration of grading scale used to evaluate non-specific background staining in a PAP procedure for cy tok er ati n .....................................27 Plate 4. Comparison of routine and microwave PAP procedure for cytokeratin staining r e a c t i o n s ........................................... 34 Plate 5. Comparison of a routine PAP procedure reaction and a successful microwave r e a c t i o n .............................................43 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INTRODUCTION Statement of the Project The immunoperoxidase staining technique is a relatively new procedure which uses antibodies to d emonstrate an tigenic substances in tissues. It has become a highly regarded tool to the pathologist. veals about substances in tissues can be a great aid in the process of diagnosing disease. ever, The information it re One of its drawbacks, how is that it requires many hours to complete the pro cedure. Microwave heating has been successfully used to expedite some staining procedures other than immunoperox idase staining (1,2,3) . A project was design ed to explore the possibilities of utilizing microwave energy to sig nificantly decrease the amount of time necessary to perform this procedure. The literature does not indicate that this method has ever been attempted. Special stains r outinely used in the laboratory microscopically reveal distinct structures, substances in tissues. organisms, or It is on the basis of this type of information that pathologists confirm many disease diagnoses. The time necessary to complete some of these diagnostic staining procedures can delay treatment and be detrimental to the well- be ing of the patient. importantly, though, Most the degree of specificity of a stain 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. determines its d ia g n o s t i c value. is for a particular substance, The more specific a stain the more v alu ab le it is for indicating the p res en ce of that substance. The mechanism by w h i c h stains depict histopathology is based on chemical rea c t i o n s between the stain and substances in the tissue. Because heat increases the rate of reaction for some chemical reactions, it can be used to speed up many staining procedures. The microwave oven is a fairly inexpen sive instrument which provides heat quickly by greatly in creasing molecular m o v e m e n t in substances. Its use in speeding up i m mun ope ro xid as e staining procedures, therefore, seems practical. Literature Review A review of the li terature will be c o mp ris ed of inform ation on several subj ect s which will give a basis of under standing for this project. is used in the mi c r o w a v e other histological will be described. Microwave ra diation and how it (MW) oven will be discussed. Some procedures which ut ilize the MW oven Immunocytochemistry and how it relates to the immunoperoxidase staining procedure will be reviewed with respect to stain ing for human cytoke rat in in skin sections. Microwave Energy and H e a t i n g The source of e n e r g y for a MW oven is electromagnetic radiation char ac ter ize d by a particular w av e l e n g t h described as a microwave. El ec tromagnetic radiation is defined as Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 "energy that is transmitted through space in the form of waves" (4). The size of the wavelength determines the d i f ferent types of radiation (see Figure 1). Microwaves are characterized by wavelengths varying from lO^nm to 10®nm, as compared to visible light waves which range from 400nm to 750nm (4). The Effect of Microwave Radiation on Biological Molecules Microwaves are p r odu ce d in a MW oven by a tube called a magnetron. This occurs through the interaction of strong magnetic and electric fields (1). In general, mic rowaves are capable of interaction with dipolar molecules ways: in two 1) by imparting kinetic energy and raising temperature, and 2) by altering electric fields (5). These o ccu r because MW radiation causes d ipolar molecules to rapidly osc il lat e 180 degrees. This causes increased intramolecular and inter- molecular motion, and as a result thermal energy increases(5). This energy is not enough to alter covalent bonds, could readily interact w i t h steric bonds bonds and Van der Waals interactions) for biological function (5) . but (ie., h yd r o g e n which are es sential Dipolar water molecules and polar side chains of larger proteins are the molecules in a cell which are a ffe ct ed most by MW exposure (6). The basic principle on w h i ch this MW interaction is ba sed is: "whatever the mecha ni sms of absorption of e le ctromagnetic radiation, absorbed must be thermalized" (6). the ene rg y This means that the Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 cosmic and gamma waves X-rays ultra violet waves infrared waves micro waves radiowaves 10 wavelength Fig. 1. (nm) The electromagnetic spectrum. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. energy introduced to the cell system m u s t be accounted for, once present. It is the electrical c h a r a c t e r of the mol e cules which determines how they are a f f e c t e d by micro waves (7) . Many studies involving the exact ramifications of microwave exposure to biological m o le cul es and cells have been done. Presently, several theories exist, involve a lte ration of cell function (6,7) . all of which One theory is that MW exposure causes a change in p o lar iz ati on at the boundary of m aterials with different d i e l e c t r i c constants. In a cell the mo st likely boundary is the cell membrane. change in pol arization here alters the lipia-water trans ition in the cell, function thus affecting cell Another theory has a more structural basis. A (6). It is known that the macromolecules which make up the functioning units of the cell (eg., mitochondria and ribosomes) depend on correct p ositioning of neighboring m ol e c u l e s for sequential reactions, thus proper functioning (6). Some investigators believe MW exposure causes the rotation of molecules in a cell. Therefore, the altered position of molecules which are adjacent to the macromolecules may interfere with the normal functioning of a cell's organelles. Experiments particles, involving the effect of microwaves on in general, position in cells support the idea that molecular is altered. These studies have e s t a b lished that m icrowave exposure causes particles to form distinct patterns" "randomly arranged (7). In other words, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. electromagnetic fields cause a rearrangement of particles exposed to them. Another mechanism by which it is felt that microwaves alter cell function is based on the increased temperature in a cell after MW exposure. Taylor and Cheung proposed, "Because of the approximately 100A thickness of the cell membrane, even a small absolute difference in temperature be tween two sides of the membr ane results in a large thermal gradi en t across the cell membrane" then, (6). This gradient, affects the flow of solutes and solvents across the membrane. In this way cellular function is altered. Laboratory Use of a Microwave Oven The microwave oven has been successfully used as a heat source for other histological procedures including fixation, and metallic impregnation in some special stains (1,2,3). In reference to tissue fixation (1,2), the t i s sues to be fixed are placed in saline or formalin, then heated in M W oven which is controlled to keep the t e m p erature from rising above 62° C. of tissue proteins, and, thus, The result is co agu la tio n fixation of the tissue for histological purposes. Another procedure successfully carried out in a MW oven is metallic histological staining metallic Routinely, impregnation for stains such as methena.T.ine silver takes several hours to complete heat, (1,2,3). (8). Using MW produced this staining is done in 60 to 75 seconds with Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. results equal to or better than those of the routine method. In particular, MW processed tissue has a reduced amount of background staining (1). Some other specific procedures which have been docu mented as successful, and a comparison of their routine and MW reaction times are found in Table 1. not commomly used in the laboratory, for future use. Although these are they do hold possibility As has been stated previously, rapid pro cedures for a variety of special stains would gr ea tly aid the pathologist in makin g diagnoses more quickly. Immunocytochemistry Antibodies are a diverse group of proteins w hich are divided into five classes called immunoglobulins (Ig). though all immunoglobulins share many properties, members of each class function differently (9) . Al the They are all part of the humoral immune system of the body, and aid in the protection ag ai nst foreign substances. Humoral immunity is that aspect of the immune system which is mediated by cell products of lymphoid tissue rather than the lymphocytes themselves substance (antigen) (10). Wh en a foreign is introduced into the body, it moves throughout it and comes in contact with B lymphocytes in lymph tissue. Here the antigen programs u ncommitted B-cells to produce specific antibody proteins against it. These B-cells then mature and become plasma cells, w h ich produce the antibody molecules. Whenever the original a ntigen is Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1 A COMPARISON OF ROUTINE REACT IO N TIMES AND MICROWAVE REACTION TI MES FOR SPECIFIC STEPS IN SOME STAINING PR OC EDURES Name of stain and step involved Routine reaction times Microwave reaction time Dieterle silver impreg nation 40 min 50 sec Enzymatic acid phospha tase incubation in substrate 4 hrs 30 sec Alcian blue staining in alcian blue solution 30 min 5 0 sec Perl's iron treatment in ferrocyanide-hydrochloric acid solution 10 min 45 sec Fontana-Masson silver impregnation 34 hrs 75 sec All routine reaction times are r ep or ted here according to reference number 8. All microwave reaction times are report ed here according to reference number 2, except for the Dieterle stain, which is from reference number 3. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. introduced to the body again, the antibodies will be pr es ent to begin the chain of events which will de str oy the antigen (10) . The structure of all antibody molecules in general is similar. acids They are composed of two light chains of amino (molecular weight 22,500), amino acids and two heavy chains of (molecular wei g h t 55,000) (see Figure 2). These four subunits are held together and form Y- or T-shaped molecules by disulf id e bonds, (10). The end of the molecule with the light chains has terminal amino groups, while the heavy chain end terminates with carboxyl groups. Each of these ends has a specific role in immune reactions. The two "arms" of the Y, each with a part of one heavy chain and one light chain, comprise two of the three principle units of the antibody molecule. the Fab units. an antigen. These are called It is here where the antibody combines with This is also the flexible part of the molecule which allows for the shape variation; a Y may become a T. The third unit is the base of the molecule, of the carboxyl ends of the two heavy chains. the Fc unit. It is called Here antibodies can combine with other cells, such as macrophages and complement proteins, case with IgG. composed This aids as is the in the immune response to destroy foreign substances in the body. It is the varying sequences of amino acids at both the Fab and the Fc ends w h ich d e t e r mine the exact function of each (9). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ] COOH COOH C O OH COOH Key: = 1 light c hain - heavy ch ain nh COOH s s 2 - amino end - carboxyl end - disulfide bond F i g . 2. Schematic drawing of an antibody molecule, as adapted from Immunology III, Bellanti, e d . (9) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 11 As has been mentioned, molecules. there are five classes of these Each g r ou p differs in its general function; and, with in each group the Fab portions of the molecules differ to combine with d i f f er ent antigens. referred to as IgG, IgA, IgM, The five classes are IgD, and IgE. ular functions are listed in Table 2. Their partic Type G immunoglob ulins are involved in the immunoperoxidase staining re actions . Antibodies are formed against certain substances found in tumors. Then, they can be used in the immunoper oxidase staining technique as tumor markers. the nature, or origin, In this way of tumor cells can be identified. This method of staining is very specific for the particular antigens against w h i c h the immunoperoxidase antibodies are made. Cytokeratin Cytokeratin is an antigenic substance found in tumors w h ich are of epithelial origin. It is a member of a family of filamentous proteins called intermediate filaments, which are characterized by a diameter of 8 to lOnm. They function as part of the cytosk el eto n of eukaryotic cells (11,12). These intermediate filaments are subdivided into five groups. Each of these is found in a particular cell type as follows: 1) cytokeratin - epi thelial cells, chymal cells, 2) vimentin - m e sen 3) de sm i n - muscle cells, 4) neurofilaments - neurons, and 5) glial filaments - astrocytes (11). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2 SOME FUNCTIONS AND PROPERTIES OF THE FIVE CLASSES OF IMMUNOGLOBULIN PROTEINS Class Functions / Properties IgG fixes complement, crosses placenta, combats mi croorganisms and toxins, combines with Fab po rti on of another antibody / most abundant, smallest IgA provides immunity in external secretory systems of the body / most abundan t Ig in sero-mucous secretions IgM fixes complement, reacts first in the line of defense against bacteremia, acts intravascularly / largest IgD surface r ec ep tor on lymphocytes, probably for ini ti ati on of immune response IgE combines w i t h mast cells and basophils / responsible for signs of atopic allergy, raised in parasit e infections adapted from Bellanti, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ed. 13 All these types c onsist of a single polype pt ide unit, except for cy t o k e r a t i n which consists of 19 polypeptides, and are e xpressed differ ent ly by d if f e r e n t ep ithelial cell types in the body soluble, (12) . The cytokeratins are all water in i n t r a c e l l u a r , and are found in g re a t e s t abundance in the cells of the epidermis and its adenexa. Here they are predominately responsible for c y t o a r c hi te ctu ral support. Not all c yto ker at in subclasses will stain w i t h the anticytokeratin antibodies in a PAP for cy t o k e r a t i n procedure. No other groups of intermediate filaments will stain pos itively with c y t o k e r a t i n pr o c e d u r e s . The possible reas on that all 19 subclasses of cyto keratin do not stain with the same a nti - c y t o k e r a t i n anti bodies is related to different molecular w eig hts of the peptides. Each of the 19 cytokeratins has a characteristic molecular we ig h t v ary ing from 37 to 68 kilodaltons. example, For c y t o ke rat in formed by cells of a squamous cell tumor has a much lower m o l ec ula r weight than the cyt ok era ti n which forms over a callus on the sole of the foot. An antibody formed against one of these may not bind to the tissue of the other due to the high degree of specifi cit y w h i c h is characteristic of immune reactions (13). The Skin The e p i th el ium of skin contains an a b un da nce of cyto keratin. Therefore, skin specimens are ideal for working with anti -c yto ke rat in antibodies. A brief o ve r v i e w of Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 14 skin structure and the location of cytokeratin be discussed. in it will Skin is composed of three di s t i n c t layers. From the surface going d ee p they are: epidermis, and subcutaneous fat dermis, (14). The epidermis contains the greatest am oun t of cyto keratin. It is made of a stratified squamous cornifying epithelium, with cells called keratinocytes predominating. The dermis is mostly connective tissue in w h ic h there are blood vessels, nerves, eccrine sweat glands. pilosebaceous-apocrine glands, and Below this is the s ub cutaneous fat which varies in thickness according to location in the body (14,15). The epidermis is compos ed of five regions of cells. These are related in a developmental/cytomorphic gradient, that is to say, the d eep est layers are immature forms of the more superficial ones. The outermost layer of corn- ified cells is formed by the maturation of the epidermal keratinocytes superficial, (14). The epidermal layers, from deep to are: 1. Stratum g er min a t i v u m - cuboidal to columnar, mitotically active cells. 2. Stratum spinosum - cells which contain elongated nuclei, and have cytoplasmic processes which att a c h to adjacent cells by forming desmosomes. 3. Stratum gra nul os um - flattened cells w h i c h contain conspicuous keratohyalin granules. 4. Stratum lucidum - closely compacted eosinophilic Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 15 layer with no nuclei, 5. found only in thick skin. Stratum corneum - many layers of flat, cornified, anuclear cells with k er a t i n being the major c o mpo ne nt (15). The cell morphology and location of these regions are seen in Figure 3. It has been immunohistochemically d o c um ent ed that, general, in cytokeratin proteins can be detected in all regions of the epidermis and in some skin adenexa including hair follicles, (16,17). sweat glands, and ducts of sebaceous glands There also seems to be an association of cy tok er atin protein with des mosomes in the epidermal cells. This suggests that c yt oke rat in may be related to cellular architecture (17). Therefore, these are the specific areas where there will be immunohistochemical positivity for cytokeratin staining. Immunoperoxidase Staining Immunoperoxidase staining is completed through a series of antigen-antibody reactions. Through these, gens can be mi cro scopically demonstrated in tissues. a nt i The value of this type of staining is becoming increasingly more apparent. Specifically, the presence of c er t a i n antigens in tissues a nd/ o r cells can lend insight into a disease entity which may be present, through cell morphology alone but not apparent (18,19,20,21,22). The ultimate goal of this technique would be to help insure proper disease diagnoses through the de t e c t i o n of Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 16 * a r a t e t : S- corneum S. lucidum S. granulosum S . spinosum S - germinativum B Fig. 3. Cell layers of skin. A. cell layers which reflect stages of keratinization - the vertically oriented columna r basal cells are transformed into horizontally aligned thin c orn ifi ed cells, taken from Moschella and Hurley (20) . B. section throu gh skin of human shoulder, taken from Bloom and Fawcett (15). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. various antigens. The pot e n t i a l based in the literature (20,21). on which a diagnosis is based tumor cells. for this seems to be solidly For instance, one aspect is the site of origin of In cases of p o o r l y differentiated malignant neoplasms the origin or p r i m a r y site of growth of cells may be very difficult to establish. Identification of specific substances in these cells may help make this more clear. For example, the difference b e t w e e n poorly differentiated lymphomas and undifferentiated carcinomas may not be apparent through cell morphology. But, in the cells would indicate it would, the presence of cytokeratin is of epithelial origin, therefore be an e p i t h e l i a l l y derived carcinoma, not a mesenchymally derived lymphoma. antigen w hich can be detected and and Cytokeratin is an th rough immunoperoxidase staining in all epithelial t i s s u e s (23). Peroxidase-Antiperoxidase Staining for Cytokeratin The peroxidase-an tip er oxi das e (PAP) method of i m m u n o peroxidase staining is an e x t r e m e l y sensitive and specific technique. Antibodies d i r e c t e d towards a variety of human antigens including hormones, globulins, viruses, proteins, immu no and enzymes are c o m m e r c i a l l y available They can be used on routinely frozen sections, In general, smears, (24). fixed paraffin sections, or c y t o s p i n preparations. the procedure consists of a series of antigen-antibody reactions, in which an antibody molecule Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 18 containing an enzyme is attached to antigenic sites tissues. Then a substrate solution is added. enzyme and substrate react, in When the a chromogen is formed, and the antigenic sites in the tissue become microscopically visible through the presence of the color. time, At the same the morphologic detail of the tissue section is p r e served. The antibodies used in the process are prepared beforehand by the injection of antigens into laboratory animals, eg., rabbits or swine, which produce IgG molecules against the specific antigens (18). The antibodies are separated from the animal blood and are purified. The PAP procedure, (see p. 62) as described in a DAKO PAP kit is as follows; 1. Hydrogen peroxide is applied to the tissue for 5 minutes. This reacts with any endogenous peroxidase present in the tissue, and will prevent it from reacting with the substrate later. This makes the procedure more specific for the anti-cytokeratin antigen 2. Next, (24). serum from a normal swine been given the antigen) for 20 minutes. (one which has not is applied to the tissue sections This reacts with the non-specific binding sites in the tissue, such as are found in collagen. This is necessary so the primary antibody in the next step does not cross-react with the non-specific binding sites, interpretation difficult making (24). 3. The primary antibody, rabbit anti-cytokeratin, applied to the sections for 30 minutes. The antibody Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. is 19 reacts and becomes chemically bo und to the antigenic binding sites of cytokeratin (24). 4. Swine anti-rabbit link serum is then placed on the tissue for 30 minutes. This combines with the primary anti -cytokeratin already bound in the tissue. It acts as a link between the primary a n ti -cy to ker at in and the peroxidaseantiperoxidase, which comes in the next step. This normal serum must be from the same species as the link serum in step 2. Because antibodies have two binding sites, the link is added in excess to ensure that at least one site on the antibody remains free to bind the PAP (24). 5. Peroxidase-antiperoxidase reagent is applied to the tissue next for 30 minutes. This a pe ntagonally shaped molecule made of an antigen-a nti bod y complex. it consists of three h o r s e r a d i s h IgG molecules, Specifically peroxidase molecules and two as is seen in Figure 4. The peroxidase is a g lyc opr ot ein w it h an 18% carbo hydrate content. Because this g lycoprotein portion is not necessary for the enzymatic activity wit h the peroxidase, it can be oxidized with p e r i oda te to form aldehyde groups. It is these groups which are c ou ple d to the amino acid groups of the IgG to form the PAP molecule (18). This molecul e binds with the link antibody w h e n it is applied to the tissue. 6. Lastly, aminobenzadine a substrate solution, prepared with 3,3'-di- (DAB), hydrog en peroxide, and phosphate Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 20 Fab Fab Fc Fc IgG IgG Fab Fab P = peroxidase molecule F i g . 4. Schematic diagram of a peroxidase-antiperoxidase molecule, taken from Sternberger (18). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 21 buffered saline utes. (PBS) is placed on the tissue for 10 m i n The peroxidase catalyzes the release of oxygen from hydrogen peroxide, with subsequent oxidation of DAB. results This in the localized precipitation of an insoluble polymer that is brown (the chromogen) (20). As can be seen in Figure 5, wherever c yt oke rat in is present in a tissue section, a brown color will occur. This enables one to m icroscopically observe the presence of this antigen in cells. Rationale The increasing widespread use of the immunoperoxidase method of staining warrants the need for improving the efficiency of the technique. One of the major problems of the procedure is the amount of time needed for its c o m pletion. If the immunoperoxidase procedure could be carried out in less time than the several hours now r e quired, it would be an even more valuable tool for the p a t h ologist. This is the primary reason why this project will attempt to utilize microwave radiation produced in a m i crowave oven to decrease the time required for the immunoperoxidase reactions. A n oth er reason deals with the value of this staining procedure. Immunoperoxidase staining can be used to help identify the primary site or cell origin of tumor cells. This information is critical nosis, for proper diagnosis, prog and treatment of c ertain neoplastic diseases. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 22 BROWN substrate DAB PAP enzyme SWAR link a nti-cyto keratin primary antibody cytokeratin antigen A Fig. B C D 5. Schematic Dia gr am of Peroxiaase-Antiperoxidase Procedure, adapted from Taylor (19). A. application of primary antibody to antigen B. ap plication of swine anti-rabbit link C. appli ca tio n of PAP enzyme complex D. c ompleted procedure with DAB added KEY: jtk ▲ ^ cytokeratin antigen primary antibody swine anti-rabbit link antibody peroxidase-antiperoxidase complex BROWN diaminobenzadine Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 23 Therefore, this p ro c e d u r e has great potential for use in surgical pathology. The practicality of the MW oven as a pi ece of laboratory equipment is another reas on this project w o u l d be reason able to perform. to purchase, It is a relatively inexpensive instrument and it is convienient to use and store. For these reasons the MW ov en would be readily ad a p t a b l e to a laboratory setting. The peroxidase-antiperoxidase method for cytokeratin localization was c h o s e n as the standard for this project for several reasons. It has been well doc u m e n t e d in the literature as giving g ood results by the routine method, wh ich is attributed to the stability of c y t o k e r a t i n as an antigen (25,26). Its antigenicity seems to w i t h s t a n d histological processing very well. cytokeratin proteins, Skin is a b u n d a n t in and is readily accessible in large enough quantity to en su r e constancy for m ul tip le test trials. Because of all the above stated advantages of the immunoperoxidase m e t h o d of staining for cy t o k e r a t i n in the skin, this project w i l l use the MW oven to a tt e m p t to reduce the amount of time required for this procedure. The overall reasoning behi nd this concept is b a s ed on the premise that expediting this staining p rocedure may make it a more useful m et h o d to detect medically impo rta nt substances in tissue, particularly those found in tumors. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MA TER IAL S AND METHODS Using sections of hu man thigh skin (6 microns thick) from randomly selected surgical specimens obtained at the West Haven Veterans A d m i n i s t r a t i o n Medical Center, peroxidase staining for c y t o k e r a t i n was done A Sears Kenmore micr owa ve oven immuno (see Plate 1) . (model number 5658758410) with an output of 650 w a t t s was used in an attempt to r e duce the amount of time for staining. using maximum MW power This work was done ("Hi" setting on MW oven), and v a r y ing time only. The quality of all slides was evaluated with respect tointensity and localiz ati on of the color change produc ed by the enzyme-substrate reaction. Staining of the specific areas believed to co nt a i n cytokeratin, eg., epidermis, were graded on a scale of 0 to +4, with 0 representing no stain reaction and +4 representing the most intense Non-specific background areas, in terms of none overstained (N), s l i g h t (I.O.) (see Plate 2). eg., dermis, were evalu at ed (S), moderate (see Plate 3). (M), or intensely Using this system the ideal MW exposure time wa s selected for each tested step. Preliminary studies were done to determine the t o l e r ance of the skin sections and immunoperoxidase reagents to MW exposure. first, Based on these, the remaining studies tested, the individual tol e r a n c e of each selected PAP reagent; and second, the combi ned tolerance of all the 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Plate 1. Human skin from thigh stained by routine PAP procedure for cytokeratin (see appendix I and II). A. Negative control stained without anti- cytokeratin (0 on grading scale) (40X). B. positive control (+3 on grading scale)(40X). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Plate 2. Illustration of grading scale used to evaluate specific cytokeratin stain reaction in a PAP procedure. Human skin from thigh. A. +1, B. +2, C. +3, D. +4 (40X). For illustration of stain quality equal to 0, refer to Plate 1. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 27 Plate 3. Illustration of grading scale used to evaluate non specific bac kground staining reactions in a PAP procedure for c ytokeratin (note the skin d e r m i s ) . A. none (N), B. slight (S), C. moderate (M) and, D. intensely overstained (I.O.). (40X). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 28 selected PAP reagents to acc um ula ted MW exposure in the same PAP procedure. swine serum The s e le cte d reagents were: (step 3), primary antibody anti-rabbit link serum (step 5), normal (step 4), swine and PAP complex (step 6). These four reagents were c h o s e n for MW treatment b ecause they require the longest incubations. Testing individual serum r eag en t tolerance to M W e x p o sure was done by adding brief M W exposures to an o t h erw ise routine PAP procedure. First, normal serum was ap pli ed to five skin sections at step 3 in a PAP procedure. T hree of these five slides were irra di ate d one at a time in the MW oven. The length of irradiation time, chosen according to the preliminary tests, was 15, removal from the MW oven, 20, and 25 seconds. t hese three slides, After as w el l as the two non-microwaved slides, w e r e continued through a routine PAP procedure for cytokeratin. The two slides not treated in the MW oven were used as p os it ive and negative controls. This same procedure was done three more times, once each for: anti-cytokeratin serum, and PAP complex. the 0 to +4 scale. swine anti-rabbit link serum, These 12 slides were graded according to This d e t e r m i n e d if each of the four reagents was affected in any w a y by the three MW exposu re times as well as the optimum M W treatment time for eac h of the four r e a g e n t s . Using these optimum MW e xpo sur e treatment times, it could be determined whether the immune reactions of these four reagents could be com p l e t e d using MW treatment, with Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 29 no additional room temperature incubation. of the four PAP serum reagents w as again in the MW oven. stained, To do this e a c h individually tested Sections of skin were routinely PAP except for the step at w h i c h a particular r e a g e n t was to be tested. First, normal serum was tested. Three slides of s k i n wer e rout ine ly brought to step 3 in a routine PAP procedure. Then two were continued through the process for use as p o s itive and negative controls. T h e one remaining slide was irra diated in the MW oven wit h n orm al swine serum on it for the o p t i m u m time chosen from pre v i o u s wo r k seconds). (15, 20, or 25 No additional t r e a t m e n t at room temperature w a s done after this. Instead, the slide was immediately c o n tinued through the rest of the PAP procedure. showed positive results, If this slide it w o u l d be known that the n o r m a l serum reacted properly while u n d e r M W treatment, shorter du ration than is rou t i n e l y used. but for Similar te ch niques were used to individually test the reactivity of the p r i m a r y antibody, the swine an ti-rabbit link serum, and the PAP complex. The next part of the p r o j e c t was to perform a c o m p l e t e PAP procedure using MW t re at men t at all of the four se lec t e d steps. A PAP procedure was done, appropriately s u b s t i t u t ing M W treatment for routine r o o m temperature tr eatment a t the p ro per point in the p r oce du re for all four of the involved reagents. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The established procedure of reduced time was then used to stain five surgical cases which were routinely determined to be cytokeratin positive by staff pathologists at the West Haven VA Medical Center. Also, one standard positive case, already shown to give good quality MW s tai n ing results, and one case known to be cytokeratin negative were stained by the same MW procedure for use as positive and negative controls, respectively. All sections were then evaluated for quality using the previously described grading systems. These were also c om par ed to corresponding routinely PAP stained sections. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS Effects of Microwave Treatment on Individual PAP Serum Reagents Physical Tolerance of Tissue Sections and PAP Serum Reagents to Microwave Exposure These tests determined that 1 . 3 - 5 drops of water on a slide in a moist chamber would not evaporate un de r 650 watts MW exposure for up to 45 seconds; 2. normal swine serum, used as a representative for all the PAP reagents, could withstand this degree of MW exposure for up to 30 seconds before coagulation of the serum occurred/ tissue morphology, and 3. eval uat ed after routine H&E staining, was not altered by a 30 second MW exposure of serum cov er ed tis sue sections in a moist chamber. Individual Microwave Irradiation of Serum PAP Reagents with Subsequent Routine Incubations After 15, 20, or 25 seconds M W exposure, plus sequent routine incubation at room temperature, the sub all four PAP reagents retained their abilities to function properly (see Table 3). This was indicated by positive PAP staining for cytokeratin which was of equal or better q u a l it y than the non-microwaved control. In general, the longer MW exposures produced positive PAP staining of better qual ity than the shorter exposures. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 32 Table 3 Individual Microw av e Irradiation of S e r u m PAP Reagents with Subsequent Routine Incubations Reagent MW exposure time (sec)*. Resulting s t a i n r e a c t i o n s ♦♦ cytokeratin ba c k g r o u n d 15 +1 N 20 +1 N 25 +2 N 15 +3 N 20 +4 N 25 -t-3 N 15 +2 N 20 +2 N 25 +3 N 15 +2 N peroxidase-anti peroxidase serum 20 +2 N 25 +3 N positive control 0 +3 N negative control 0 0 N normal swine serum anti-cytokeratin serum swine anti-rabbit link serum ♦Immediately after the indicated MW exposures, the sections were left to incubate at room temperature as follows: normal swine serum - 20 min., anti-cytokeratin - 30 min., swine anti-rabbit link serum - 30 m i n . , and PAP c o m p l e x 30 min. ♦♦Results for specific areas believed to contain cyt o k e r a t i n are reported on a scale of 0 to +4, with 0 re pr ese n t i n g no stain reaction and +4 representing the most intense staining. Results for non-specific background areas of t i s s u e are reported in terms of none (N) , slight (S) , m o d e r a t e (M) , or intensely o verstained (I.O.). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 33 MW treatment of the primary antibody significantly enhanced the intensity of PAP staining of cytokeratin-containing cells, without significant increase of non-specific b a c k ground staining in any of the sections (see Plate 4). Microwave Irradiation of PAP Serum Reagents Without Routine Incubations Individual testing of each PAP serum reagent for its proper function after the brief MW treatment times revealed that the normal swine serum step and the swine anti-rabbit link serum could be successfully completed in the MW oven in 25 seconds (see Table 4). serum step required 30 seconds primary antibody step, The peroxidase-antiperoxidase (see Tables 4 and 5) . The after several different variations of brief MW treatment, was shown to give the best results by an initial 20 second MW exposure, immediately followed by incubation for 30 minutes at room temperature. The various methods used to shorten this step and a descrip tio n of the results are outlined in Table 6. Effects of M icrowave Treatment on Combinations of PAP S eru m Reagent Steps Co mbination of Microwave Treatments Which Were Shown to Give Positive Results Individually The steps which were ex pediated by MW treatment Tables 3,4,5) were combined in one procedure (see Table Three separate trials of the procedure were run. sections showed positivity (see The in the appropriate cells of Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 7) . 34 Plate 4 Com par is on of routine and micr owa ve PAP procedure for cytoker ati n stain reactions. Human skin from thigh. A. stained by routine PAP procedure (+3 reaction) . (40X). B. stained by adding a 20 second MW exposure to only the anti-cytokeratin step in a PAP procedure (+4 reaction, as indicated in T able 3) (40X). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 35 Table 4 MICROWAVE IRRADIATION OF PAP SERUM REAGENTS WI TH O U T ROUTINE INCUBATIONS Reagent MW exposure time (s e c )♦ Resulting stain re a c t i o n ^ ♦ cytokeratin background normal swine serum 25 +3 N anticytokeratin serum 20 +1 N swine anti-rabbit link serum 25 +3 N 25 +2 N PAP complex ♦The three reagents not being tested at a pa rt icular step were routinely used in the PAP procedure as follows: normal swine serum - 20 min., anti-cytokeratin se rum - 30 min., swine anti-rabbit link serum - 30 min., and PAP complex - 3 0 min. at room temperature. ♦♦Results for specific areas believed to contain cytokeratin are reported on a scale of 0 to +4, with 0 represe nt ing no stain reaction and +4 representing the most intense s t ai n ing . Results for non-specific background areas of tissue are reported in terms of none (N), slight (S), m od era te (M), or intensely overstained (I.O.). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 36 Table 5 FURTHER MI C R O W A V E TESTING OF ANTI-CYTOKERATIN AND PEROXIDASE-ANTIPEROXIDASE REAGENTS Reagent MW exposure time (sec)* Resulting stain reaction* * cytokeratin anti-cyokeratin serum peroxidaseantiperoxidase serum background N 30 +1 40 +4 1 .0. 50 +4 1 .0. 30 +3 N 40 0 N 50 0 N *Except for the reagent being tested, all ot her steps in the PAP procedure were done routinely as follows: normal swine serum - 20 min. at room temp., anti-cytokeratin serum - 30 min. at room temp., swine anti-rabbit link serum - 30 min. at room temp., and PAP complex - 30 min. at room temp. **Results for specific areas believed to contain cytokeratin are reported on a scale of 0 to +4, with 0 representing no stain reaction and +4 representing the most intense staining. Results for n on -specific background areas of tissue are reported in terms of none (N), slight (S), moderate (M), or intensely ov e r s t a i n e d (I.O.). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 37 Table 6 METHODS USED TO REDUCE REACTION TIME OF ANTI-CYTOKERATIN REAGENT Resulting stain reaction^♦ Method used to shorten anticytokeratin step^ cytokeratin background +1 N 30 sec. MW exposure +1 N 4 0 sec. MW exposure +3 M 50 sec. MW exposure +3 M 30 sec. MW exposure, 5 min. room temp, incubation, 30 sec. MW exposure +4 30 sec. MW exposure, 10 min. room temp, incubation, 30 sec. MW exposure +4 W • 20 sec. MW exposure 30 sec. MW exposure, 15 min. room temp, incubation, 30 sec. MW exposure +4 1 .0. • O I .O. *All other steps in the procedure are carried out by the routine method, i e ., no MW exposure. ♦♦Results for specific areas believed to contain cytokeratin are reported on a scale of 0 to +4, with 0 representing no stain reaction and +4 representing the most intense staining. Results for non-specific background areas of t i s sue are reported in terms of none (N), slight (S), moderate (M), or intensely overstained (I.O.). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 38 Table 7 COMBINED MICROWAVE TR E A T M E N T PAP PROCEDURE Reagent normal swine serum anti-cytokeratin serum swine anti-rabbit link serum peroxidase-antiperoxidase serum R e action procedure* 25 sec in MW oven 20 sec in MW oven plus 30 min at room temp 25 sec in MW oven 30 sec in MW oven This was done using exposure times obtained from the opti mu m results of the previous tests. *In this procedure the above steps are carried out in the proper sequence to complete a PAP procedure. No steps are done by the routine method. Between each step slides are w ashed in PBS buffer for 10 minutes at room temperature. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 39 intensity from + 2 or +3. However, there was a slight to moderate increase in non-specific background staining. This was not as intense as in the positively stained cytokeratin-containing cells. The negative controls which utilized normal serum at the primary antibody step, dis played evenly distrib ut ed slight to moderate n on -specific staining over the entire section in each of these trials. These particular sections were also MW treated. The cyto- keratin sites did not stain more intensely than the b a c k ground in these same sections (see Table 8). Microwave Treatment of All PAP Serum Reagents Except the Primary Antibody When MW exposure of the anti-cytokeratin serum was omitted (see Table 9), all cells containing cyt oke ra tin displayed a positive reaction of +3 intensity. background staining was still present, degree. Althoug h it was to a lesser Two of the negative control slides, also M W treated, displayed a similar pattern of non-specific staining over the entire section, Table 10). as in the three previous trials (see Trial 2 gave the best results which are illustrated in Plate 5. Microwave Staining of Five Surgical Specimens Containing C ytokeratin Five surgical cases, previously determined to show good quality PAP reactions for cytokeratin by the routine method, were stained using MW treatment during all serum Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 40 Table 8 RESULTS OBT A I N E D USING COMBINED MICROWAVE TR E A T M E N T FOR ALL PAP SERUM REAGENTS * Trial Serum used at pri mary ant i b o d y step Resulting stain reaction^ * cytokeratin 1 immune negative control immune background +3 S N/A S +2 M N/A M +3 M N/A M 2 negative control immune 3 negative control ♦The procedure used in all three trials is: normal swine serum - 25 sec in MW oven, anti-cytokeratin s erum - 20 sec in MW oven immediately followed by 30 min room temp incubation, swine anti-rabbit link serum - 25 sec in MW oven, and PAP c o m p l e x - 30 sec in MW oven (see Table 7). ♦♦Results for specific areas believed to contain c y t o k e r atin are reported on a scale of 0 to +4, with 0 repre senting no stain reaction and +4 representing the most intense staining. N/A = nonapplicable because both specific and non-specific areas of tissue stained the same. Results for non-specific background areas of tissue are reported in terms of none (N), slight (S), m o der at e (M), or intensely overst ain ed (I.O.). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 41 Table 9 COMBINED MICROW AV E TREATMENT PAP PROCEDURE* FOR AL L STEPS EXCEPT THE PRIMARY ANTIBOD Y STEP Reagent normal swine serum Reaction procedure* 25 sec in MW oven anti-cytokeratin serum 30 min at room temp swine anti-rabbit link serum 25 sec in MW oven peroxidase-antiperoxidase serum 30 sec in MW oven ♦This procedure is modifie d from the initial procedure (Table 7) used to attempt a reduction of non-specific background staining. ♦♦The above steps are ca rr ied out in proper sequence to complete a PAP procedure. Between each step slides are wash e d in PBS buffer for 10 min at room temperature. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. r 42 Table 10 RESULTS OBTAINED USING COMBINED MICROWAVE TREATMENT* FOR ALL PAP REAGENTS EXCEPT ANTI-CYTOKERATIN ■ Trial Serum used at p r i mary antibody step ■ ■ ■ ■ — cytokeratin immune 2 immune negative control immune ■ background +3 S N/A S +3 N N/A N -*-3 M N/A M 1 negative control ~ Resulting stain reaction* * 3 negative control *This procedure, used for all three trials, is: normal swine serum - 25 sec in MW oven, anti-cytokeratin serum - 30 min at room temp, swine anti-rabbit link serum - 25 sec in MW oven, and PAP complex - 30 sec in MW oven (see Table 9). **Results for specific areas believed to contain c ytokeratin are reported on a scale of 0 to +4, with 0 representing no stain reaction and +4 representing the most intense staining. N/A = nonapplicable because both specific and non-specific areas of the tissue stained the same. Results for non-specific areas of the tissue are reported in terms of none (N) , slight (S), moderate (M), or intensely overstained (I.O.). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. r 43 Plate 5. Comparison of routine PAP procedure reaction and successful microwave exposed PAP procedure reaction. Human skin from thigh. A. stained by routine PAP procedure (+3 react ion ). (4 0 X ) . B. stained by using MW treatment only for all steps except ant i-cytokeratin (see Table 9 for this procedure; also a +3 reaction) (40X). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. applications except that of the primary a nt ib ody 9). One negative control specimen to be cytokeratin negative) (see Table (previously determined and the standard s k i n positive control were also stained in this manner. The PAP MW reaction results were similar for each case t e s t e d Table 11). In general, positive staining, (see the procedure produced good quality but non-specific background present in most sections. staining was In three of the five test cases (A , B ,C ) the non-specific background staining w a s not as intense as the specific in two cases (D,E) staining of cytokeratin. However, no diff er enc e in intensity w a s seen between specific and non-specific staining. When normal serum case, (negative control) was u sed two of the six c ases had no non-specific for each staining. The four remaining cases showed slight to mo d e r a t e overall staining. In two of these four cases there was no d i f ference between staining of sections on which the primary antibody serum (anti-cytokeratin) was used, and secrions on which normal serum was used in place of anti-cytokeratin. Both the negative c ontrol sections, one stained with anti- cytokeratin serum and one stained with normal not have shown any positivity; tain cytokeratin. However, serum, should because they did not c o n there was evenly di s t r i b u t e d non-specific staining o v e r the entire section in both of these, as is shown in T a b l e 11. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 45 Table 11 COMPARISON OF ROUTINE AND MICROWAVE STAINING OF SPECIMENS KNOWN TO C O N T A I N CYTOKERATIN* Routine PAP reaction results Case positive immune serum MW PAP reaction results negative control serum positive immune serum negative control serum CK & BG CK BG CK & BG CK** BG** A +3 N N +2 S S B +3 N N +3 S N C +3 N N +3 S S D +3 N N N/A M M E +3 N N N/A M M positive control negative control +3 N N +3 N N 0 N N N/A S S ♦Determined by staff pathologists at the West Haven VA Medical Center. **CK = cytokeratin and BG = background Results for specific areas believed to contain c y t o k era tin are reported on a scale of 0 to +4, with 0 representing no stain reaction and +4 representing the most intense staining. N/A = nonapplicable because both specific and non-specific areas of the tissue stained the same. Results for non-specific areas of the tissue are reported in terms of none (N), slight (S), moderate (M), or intensely overstained (I.O.). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. D IS C U S S IO N Data Analysis The purpose of this project was utilization of the microwave oven to improve the immunoperoxidase staining technique. This was to be accomplished by decreasing the amount of time necessary to complete the procedure without sacrificing the quality of the end product. First, normal serum on a section of skin from the thigh was tested for MW tolerance. This established a maximum exposure time of approximately 30 seconds. This determination was based on whether the serum dried on the slide as well as whether the microscopic morphology of the e x posed skin sections remained unchanged. Microscopic ex am ina tio n of H&E stained exposed skin sections verified the latter. Normal serum was chosen as a representative of the four PAP reagents because they are all serum based. The only difference between reagents is that each contains d i f fer en t specific antibodies. Based on the time established above, the functional tolerance to MW exposure of all four PAP serum reagents was tested next. Each reagent was MW treated separately, and then allowed to incubate at room temperature for the routine time indicated in Appendix II. The quality of staining in each irradiated tissue section indicated if the 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 47 particular reagent being tested functioned properly. Table 3 indicates that at least one section tested for each reagent produced good quality staining, tivity was not destroyed. thus reagent reac The successful MW exposure times for all the PAP reagents were: 20 seconds for an ti-cytoker atin serum, and 25 seconds for normal serum, rabbit link serum, swine a n t i and peroxidase-antiperoxidase serum. These findings are important in two ways. First, it has been conclusively determined that for the MW exposures listed above, MW radiation has no harmful effect on reagents or tissues. tinued. Second, Thus, further testing can be c o n as is indicated in Table 3 and is seen in Plate 4, the 20 second MW exposure of anti-cytokeratin, prior to the routine 30 minute incubation at room t e m p erature, greatly enhances the stain quality. The intensity of staining in this particular skin section was even much greater than in the positive n o n microwaved control. It is important to note the stain positivity is localized and limited to those structures in the skin which are believed to contain cytokeratin, the epidermis. ing. eg., There is no non-specific background stain So, for this particular serum step, the added 20 second MW exposure improves the immunoperoxidase stain quality. This enhanced qua lit y staining is probably related to the heat produced by the microwaves during irradiation treatment. When the slides with the serum were removed Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 48 from the MW oven after each trial, were warm. it was noted that they It has been long established that the rate of most chemical reactions increases as temperature rises (27). One may speculate that the heat produced by the MW oven during the 20 second M W exposure increased the moveme nt of the anti-cytokeratin molecules. This increases their kinetic energy and allows them to bind to more antigenic sites in the tissue, w h i c h results in more intense staining. The question arises here whether the radiation from the MW oven produces he at which then leads to increased kinetic energy of the irradiated molecules, kinetic energy increases production. or w hether first, which then leads to heat Because the PAP stain reaction occurring after a 20 second MW ex po s u r e of anti-cytokeratin serum was appropriately locali ze d in the tissue sections, there is no reason to believe that the antibodies or antigens have been damaged, as m i g h t be induced by heat. Therefore, it may be surmised that the MW irradiation caused increased molecular kinetics, which, rate. in turn, increased the reaction This led to i ncreased stain intensity. of heat produced, then, The majority probably came after increased kinetic energy. The results ob ta i n e d in the next part of the project (outlined in Tables 4 an d 5) determined that the PAP serum reactions, except the an ti-cytokeratin step, could indeed be completed with MW exposure. This was concluded because except for the step at w hich a particular reagent was being Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 49 tested, the remainder of the procedure was done routinely. A positively stained section meant that all reactions procedure were properly completed. in the It is likely that in creased molecular movement along with heat induced by the microwaves are responsible for the reaction completeness which occurred at each serum reaction step. This allowed for sufficient binding of antibody to antigen. By the end of the procedure there must be sufficient amount of p e r o x idase enzyme present on the tissue to react with enough DAB substrate to give good quality staining. The positive staining is localized at the proper sites ir. the tissue, where cytokeratin is be li e v e d to be present. ie., Thus far the procedure has not lost its specificity as a result of MW exposure. As has been mentioned, the anti-cytokeratin step does not appear to be co mpleted using MW exposures 4,5,6). (see Tables For MW exposures of 30 seconds or less, ing quality of tissue sections resulted. weak and focally d istributed poor stain The staining was (see Table 6). These results could be interpreted to mean the reaction was not completed due to insufficient molec ula r agitation produced by the microwaves. It is not believed that the incomplete reaction of the anti-cytokeratin step is due to destruction of the a n t i bodies in the reagent or the antigens in the skin by the microwaves. This is because this reaction was successfully completed when sections and serum were irradiated for 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 50 seconds, minutes then left to incubate at room temperature for 30 (see Table 3). Here, the MW exposure did not result in the destruction of any necessary components of the reaction. For MW exposures of greater than 30 seconds, whether as a continuous MW exposure or alternating with room t em p erature incubations, poor staining guality occurs. In comparison to a 30 second or less MW exposure, which re sulted in focal light staining, these longer MW exposures resulted in intense n on- specific overstaining. Alternating MW exposures with room temperature incubations should have kept heat production to a minimim. removal from the MW oven, But, even after increased kinetic energy of the substances on the slide allowed the excess heat to remain. The majority of heat produced by the microwaves, then, most likely occurs after kinetic energy has been increased. Here, 30 seconds seems to be the critical point at which heat becomes excessive. It is believed that heat is re sponsible for this intense non-specific overstaining by altering molecular c onf ormation of the exposed proteins. Specifically, the non-specific overstaining may pos sibly indicate that the binding sites on either the antigen or the antibody are altered in such a way that the primary antibody attaches to many non-specific binding sites over the entire tissue section, That is, protein structure, not just cytokeratin. probably molecular conformation, may be altered by the MW induced heat. This may occur on Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 51 non-specific tissue proteins, thus destroying the tissue and allowing the Fc p ortion of the anti-cytokeratin to bind anywhere. On the other hand, the structure of the Fc site itself may be altered, Additionally, thereby making it non-specific. increased MW radiation continues to increase molecular movement. The combination of altered molecular structure and increased molecular activity probably causes the excessive non-specific overstaining which has been observed. At this point it was concluded that the reaction times for only the normal serum, swine anti-rabbit link serum, and peroxidase-antiperoxidase serum may be possibly reduced in one PAP procedure. The primary antibody reaction would best be done by a 20 second MW exposure followed by a 30 minute room temperature incubation. This would still greatly reduce the am ou n t of time required for the entire PAP procedure. This s h o rt ene d procedure is outlined in Table 7. The stain reactions following this procedure (see Table 8) were not of equal quality to slides stained by the routine method. positively, A l t h o u g h the cytokeratin did stain more non-specific background staining than was present in the routinely stained control sections was also present. This may m a ke interpretation of an unknown tissue specimen uncertain. As would be expected, the negative control sections, which were also MW trea ted and stained using normal Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 52 non-immune serum in place of anti-cytokeratin serum, the same non-specific staining. In these sections, showed though, cytokeratin was not more intensely stained than the b a c k ground. This means that the DAB was able to bind to the tissue even though no anti-cyt oke ra tin was present. It is believed that the mec han ism for the non-specific staining observed is probably due in part to the c o n s e quences of increased kinetic energy and heat production which have already been discu sse d (see pages 50-51). But, because each reagent was irradiated for only 30 seconds or less; and, each slide was im mediately cooled in a room temperature PBS bath, increased kinetic energy and heat can not be the only factors involved. Therefore, c o m b i n ations of individually successful MW exposures done in one PAP procedure must lead to an accumulation of radiation for each section stained. This c u m u la ti ve effect of MW rad iation must go beyond molecular agitation and heat p r o duction. Some other unknown factor could be at blame for the non-specific staining. The non-specific staining could occur at any point in the procedure. It is probably the swine anti-rabbit link serum or the peroxidase-antiperoxidase serum, or both, which non-specifically bind to the tissue. occur later in the procedure, The normal swine serum step after 25 seconds MW exposure These steps after accumulated MW exposure. (step 1) functioned properly (see Table 4). Therefore, Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 53 the non-specificity is not due to unbound endogenous non specific binding sites in the tissue. Endogenous tissue enzymes could be responsible for some of the non-specific staining observed. In the PAP procedure hydrogen peroxide reacts with endogenous tissue peroxidase (see Appendix II). This eliminates an inap propriate reaction with DAB later. The accumulation of MW radiation may increase the activity of endogenous peroxidase left unbound by hydrogen peroxide. The radiation may also change the structure of other endogenous enzymes, rendering them capable of a reaction with DAB. Next, a combined MW procedure, with no MW exposure at the primary antibody step, was done to attempt elimination of the non-specific staining (see Table 9). This, however, yielded results similar to those previously obtained Table 10). (see The non-specific staining in the positive and negative controls was of slightly lesser intensity, or in one case, not present at all (see Plate 5). The 20 second decrease in cumulative MW exposure most likely accounts for this. Reproducibility of the above results was tested using five surgical cases known to contain cytokeratin. stain reactions The (see Table 11) were inconsistent and not of diagnostic quality. Generally, not enough of a di f ference between slides stained with immune serum and those stained with normal, non-immune serum was evident. The amount of cumulative MW radiation used here appeared to be Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 54 incompatible with succe ssf ul completion of the immunoperoxidase reactions. Microwave r ad i a t i o n can be used to im pro ve the immunoperoxidase technique; not by decreasing the am ou n t of time required for the procedure, intensity. but by enha nci ng the staining In this w a y limited MW exposure improves the quality of obtainable results acity, (see Plate 4). In this cap the goal of u t i l i z i n g the MW oven to improve the PAP technique has been met. Although this is the most positive significant finding r es u l t i n g from this project, all the data holds its own pa r t i c u l a r value. The enhanced q u a l i t y of staining increases the ease with wh ich immunoperoxidase stained slides are evaluated. This makes the technique mo re valuable as a di a g n o s t i c as well as a research tool for pathology. Also, the M W exposu re may accentuate otherwise undetectable specific posi tiv it y on tissues. In these ways, the results of this pr oject can be applied to the general field of pathology. The remaining d a t a holds most significance for future w o r k on this topic. The fact that each in dividual PAP serum reagent is not des t r o y e d by a limited amo unt of MW r a d iation, is of p ar ticular importance. This i nformation implies there may still be a way to use the M W o ven to decrease the time r e qui re d for immunoperoxidase staining. As long as the specific reactivity of the a nt ige ns and antibodies can be maintained, work towards this goal can be continued. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It would be worthwhile to continue the efforts ocgun here to shorten the PAP procedure. The first suggestion for follow up work on this project is to develop a way to monitor the temperature of the reagents during MW treatment This capability would allow for the use of lower MW setting In this way the antigens and antibodies could be exposed to a lesser degree of radiation. The exposure, however, might be enough to successfully complete the required immune reactions. Determination of the exact point at which MW radiation becomes excessive is another suggestion for follow up work. If this point at wTiich the tissues and/or the reagents b e come destroyed was known, possioly still be used, three steps. a comoined MW7 procedure could even if only for the first two or The total procedure time could still be si g nificantly reduced. Another approach may separate the increased reactivity from the detrimental loss of specificity. were Kept cool, If the slides possibly on ice, during the MW exposure, denaturation of proteins may be reduced. This is assuming that the non-specificity was a result of heat and not a direct effect of microwaves. Elimination of non-specific background staining would possibly improve the q uality of the stain reaction results of a PAP MW procedure. as trypsin and pepsin, Recently, proteolytic enzymes, have been successfully used in routine PAP procedures for this purpose (27,28). It has Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. such 56 been found that such enzyme treatment antigens, thereby making "unmasks" tissue immunohistochemical reactions more specfic by reducing non-specific background staining. The mechanism by which tnis occurs is thought to be related to enzymatic activity which 1) destroys ground substance pro tein molecules that are in connective tissue (27), or 2)in- creases cell and tissue permeability to the macromolecules that are in the PAP reagents sibility of the antigens treatment, (28). This increases acces in the cells. Such an enzyme then, wo uld be worthwhile to try when performing a MW PAP p r o c e d u r e . Overall Conclusions The goal of this project was to dev e l o p an immunoper oxidase staining technique which implements a MW oven, and requires less time than is routinely necessary. Several preliminary tests we re performed in order to provide the basic information required to formulate the final method which was developed (see Table 9). These tests involved using the MW oven in several PAP procedures with careful evaluation of each slide stained. The conclusions drawn from the sequence of tests done in this project are: 1. Individual MW irradiation of skin sections covered with PAP antibody and enzyme reagents for 30 seconds or less does not destroy the reactivity of antigens or anti bodies required for good quality PAP staining. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. J / 2. Cumulative MW expos ure s in one PAP procedure Joes appear to destroy the involved PAP reagents or the tissues, or both, resulting in non-specific staining along with increased intensity. 3. The probable cause of the unfavorable staining results is related to the increased Kinetic energ y of the exposed substances, and the subsequent heat production, as well as possible u nk n o w n factors. 4. The MW oven can be used to greatly e nhance tire stain intensity resulting f r om a routine PAP procedure for cyto keratin. This is done by a 20 second MW exposure of the tissue section covered with the anti-cytokeratin serum prior to routine room temperature incupation. Increased molecular movement duri ng this brief MW exposure leads to increased stain intensity without loss of specificity. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES 1. 2. Brinn N.: Rapid Me ta l l i c Histological Staining Using the Microwave Oven. Journal of Histot ec hno lo gy. 6^, 125-129 (1983). Brinn N . : Microwave Ovens for Metallic Histologic Staining: A New Concept. Duke University Medical Center Durham, North Carolina. (1984). 3. Sohn L. and Shapiro S.: Rapid Dieterle Stain with Micro wave Heating. Queens Hospital, Long Island Jewi sh- Hi llside Medical Center. Jamaica, New York. (1984). 4. Fessenden R, and Fe s s e n d e n J.: Organic C h e m i s t r y , PWS Publishers, Boston, MA, 314 (1982). 5. Login G. and Dvorak A.: Microwave Energy Fixation for Electron Microscopy. Am.J.Pathol. 120, 230-243 (1985). 6. Taylor L. and Cheung A. (ed.).: The Physical Basis of Electromagnetic Inter ac tio ns with Biological S y s t e m s , U.S. Department of Health, Education and Welfare. Rockville, MD, (1978). 7. Czerski P. et a l .: Bi ological Effects and Health Hazards of Microwave R a d i a t i o n , Polish Medical P u b lishers, Warsaw, P o l a n d ,(1974). 8. Humason G.: Animal T is s u e T e c h n i q u e s , W. H. and Company, San Francisco, CA, (1979). Freeman 9. Bellanti J . : Immunology I I I . W. B. Saunders Philadelphia, PA, (1985). Company. 10. Bellanti J. Immunology I I . W. B. Saunders Company. Philadelphia, PA, (1978). 11. Battifora H . : Mon oc lon al Antibodies in the H i sto logic Diagnosis of Cancer. Laboratory Management. 38-53 (1985). 12. Lazarides E.: Inte rm edi at e Filaments as Mechanical Integrators of Cellular Space. Nature. 283, 249-255 (1980). 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 13. DAKO Bulletin. New Tissue Staininq Kits and AntiSera for Keratin/Wide Spectrum Screening. DAKO Corporation. 22 North Milpas St., Santa Barbara, CA 93103 (1985). 14. Moschella S. and Hurley H. (ed.): D e r m a t o l o g y , vol. W. B. Saunders Company, Philadelphia, PA, (1985). 1, 15. Bloom W. and Fawcett D.: A Textbook of H i s t o l o g y , W. B. Saunders Company, Philadelphia, PA, (1975). 16. Moll M . , Werner F., Schiller D., Geiger B., Reinhard K. The Catalog of Human C y t o k e r a t i n s : Patterns of Expres sion m Normal Epithelia, Tumors and Cultured Cells. Cell. 3_1, 11-24 (1982) . 17. Schlegal R . , Schlegal S., Pinkus G.: Immunohistochemical Localization of Keratin in Normal Human Tissues. Lab. Invest. 4_2, 91-96 (1980). 18. Sternberger L . : The Unlabeled Antibody Enzyme Method of I m m u n o h i s t o c h e m i s t r y . J. Histochem. Cytochem. 1 8 , 315-333 (1970). 19. Taylor C. and BChir D.: Arch. Pathol. Lab. Med. Immunoperoxidase Techniques. 102, 113-121 (1978). 20. Mesa-Tejada R . , Pascal R., Fenoglio C.: I mm u n o p e r oxidase: A Sensitive Immunohistochemical Technique as a Special Stain in the Diagnostic Pathology L a b oratory. Hum. Pathol. S3, 313-319 (1977). 21. DeLellis R . , S ternberger L . , Mann L . , Banks P., Nakane P: Immunoperoxidase Techniques in Diagnostic Pathology. Report of a Works ho p Sponsored by the Nat:onal Cancer Institute. Am. J. Clin. Pathol. 7_1, 483-488 (1979). 22. Banks P.: Diagnostic Application of an Immunoperoxidase Method in H e m a t o p a t h o l o g y . J. Histochem. Cytochem. 2 7 , 1192-1194 (1979) . 23. Nagle R., McDaniel K., Clark V. , Payne C.: The Use cf Antikeratin Ant ibodies in the Diagnosis of Human N e o plasms Am. J. Clin. Pathol. 79_, 458-466 (1983) . 24. DAKO PAP Kit Instruction Booklet. DAKO Corporation, 22 North Milpas St., Santa Barbara, CA 93103 (1980). 25. Gabbiani G, Kapanci Y., Barazzone P., Werner F.: Immunochemical Identification of Intermediate-sized Filaments in Human Neoplastic Cells. Am. J. Pathol. 104, 206-216 (1981) . Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 60 26. Schlegal R., Schlegal S., McLeod J., Pinkus G.: Immuno peroxidase Local iza tio n of Keratin in Human Neoplasms. Am. J. Pathol. 101, 41-50 (1980). 27. Reading M . : A Diges tio n Technique for the Reduction of Background Staining in the Immunoperoxidase Method. J. Clin. Pathol. 3_0' 88-90 (1977). 28. Curran R. and Gregory J. : Demonstration of Immuno globulin in Cryos ta t and Paraffin Sections of Human Tonsil by Immunofluoresence and Immunoperoxidase Techniques. J. Clin. Pathol. 3_1, 974-983 (1978). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A P P E N D IX I Reagents Used in the PeroxidaseA n t i p ero xi das e Procedure* 1• 3% H 2 O 2 2. Normal swine serum 3. Primary antibody 4. Link antibody 5. PAP complex 6. Non-immune rabbit serum 7. DAB reagent: Add 2-3mg DAB to 2.5ml PBS, before use, add 5-8 d rops 3% H 2 O 2 8. Xylene 9. Ethanol; 10. Phosphate buffered saline 11. Harris' 100%, 95%, (negative control) immediately 80% hematoxylin 12. Ammonia water: mix 15ml of c o n e . ammonium hydroxide with 1 liter tap water 13. Acid alcohol: alcohol mix 10ml c o n e . HCl with 1 liter 70% 14. EUKITT mounting me di um (Calibrated Instruments, 731 Saw Mill River R d . Ardsale, New York 10502) ♦Reagents 1-7 obtained from DAKO Corporation, Milpas St. Santa Barbara, CA 93103. Inc. 22 North Reagents 8-14 routinely used at the West Haven V A Medica Center, West Haven, CT 06516. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A P P E N D IX I I Pero xi das e-Antiperoxidase Staining Procedure* 1. If paraffin sections are used, deparaffinize sections in 2 xylene baths. R e h ydr at e in graded alcohols, 100%, 95%, and 80%. 2. Wipe slides around tissue a n d place in mo ist chamber. Apply 2-3 drops 3% H 2 O 2 to cover tissue, incubate. Wash slides in Phosphate b u f f e r e d saline (PBS) 5 min ea. 3 min ea. 5 min 10 min 3. Place slides in moist chamber, apply normal serum to specimen and incubate. 20 min 4. Tap off excess serum and a p p l y primary antibody to s e c t i o n s , use non-immune serum as negative control, incubate. Wash slides in PBS. 30 10 mm min 5. Wipe slides around tissue. Apply link antibody to tissue sectio ns and incubate. Wash slides in PBS. 30 10 min min 6. Wipe slides around tissue, reagent, incubate. Wash slides in PBS. Apply PAP 30 min 10 min 7. Place slides in moist c h a m b e r and apply freshly prepared D i a m i n o b e n z a d i n e - H 2 0 2 r e agent to slides. Rinse slides in PBS. 10 min 10 min 8. Cou nterstain slides in Harris' hematoxylin. Wash in tap water. Rinse briefly in acid alcohol. Place slides in ammonia water. Wash in tap water. Dehydrate slides in g r a d e d alcohols: 80% 95% 3 baths 100% Place slides in two xyle ne baths. 5 min 5 min 2 dips till blue 5 min 2 dips 2 dips 2 dips ea, 2 min ea. 9. Cove rs lip with EUKITT ♦Procedure used at the West H a v e n VA Medical Cente r 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.