RETURNING MATERIALS: IV1ESI_} Place in book drop to IJBRARJES remove this checkout from “ your record. FINES win be charged if book is returned after the date *stamped below. IMMUNOSTAINING OF CANINE LYMPH NODES USING THE AVIDIN BIOTIN COMPLEX TECHNIQUE BY Steven Andrew Stefanski A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pathology 1984 ABSTRACT IMMUNOSTAINING OF CANINE LYMPH NODES USING THE AVIDIN BIOTIN COMPLEX TECHNIQUE BY Steven Andrew Stefanski Five normal canine lymph nodes were stained with a commercial avidin-biotin—peroxidase (ABC) kit, anti-canine immunoglobulin G and DT-2 (a monoclonal antibody reportedly specific for canine T lymphocytes) to determine the usefulness of these immunologic reagents in dogs. Both frozen and fixed tissue sections were utilized. The anti-canine immunoglobulin G had specific staining predominately in B cell areas, the medullary cords and the central follicular areas. The DT-2 antibody stained paracortical areas, interfollicular zones and the center of larger secondary follicles, all of which are known for T cell activity. No major modifications were necessary for adapting the ABC kit for use in canine lymphoid tissue. Dedicated to my wife, Linda, and my children, Kimberly and Matthew. ii ACKNOWLEDGEMENTS I wish to express my sincere thanks to the members of my committee, Dr. Janver Krehbiel, Dr. Robert W. Bull, Dr. Wanderley DeMendonca, and Dr. Robert Dunstan, for their expert guidance and moral support during the course of this research project. In addition, I also wish to thank Mae Sunderlin and Peggy Coffman for their expert technical assistance. Finally, I gratefully acknowledge the love, understanding, and the many sacrifices that my wife made in support of me to finish this project. H. I-‘- p. TABLE OF CONTENTS Page LIST OF TABLES 0.00.....0.0..O...OOOOOOOOOOOOOOOOOOOOOO. Vi LIST OF FIGURES O00.0..OOOOOOOOOOOOOOOOOOOOO0.0.0.0.... Vii INTRODUCTION O00....00....OOOOOOOOOOOCOOOOOCOO00.0.0.0... 1 N LITERATURE REVIEW .00..00....00.000.000.000...0.0.00.0... Embryology ......................................... Anatomy ............................................ Immun010gic zones 000......O...OOOOOOOOOOOOOOOOOOOCO ImmunOStaining MethOdS O0..OOOOOOOOOOOOOOOOOOOOOOOOO Procedural Considerations .......................... ResearCh in Dogs OOOOOOOOOOOOOOOOOOOOOOOOO0.0.0.... 1 NKOC‘ANN MATERIALS AND METHODS 0.0.00.00.00.000...00.00.000.000.. 13 Materials 0.000......O00.00....OOOOOOOOOOOCCOOOOOOO 13 Immunologic Reagents .......................... 13 Immunoperoxidase Kits ......................... 13 Other Reagents ................................ 14 Tissue OOOOOOOOOOOOOOOOIOCOOOOOOOOOOOOCOOOOOOOO 14 MethOdS O00.0.0000...0.0...OOOOOOOOOOOOOOOOOOOOOOOO 16 Preparation of Tissue Specimens ............... 16 Immunoperoxidase Staining Procedure ........... 17 Negative contrOlS OO...COOOOOOOOOOOCOOOOCOOOOOC 20 RESULTS 00....0...00...0.0.0....OOOOOOOOOOOOOOOOOOOOOOOO 2]- Analysis of Normal Lymph Nodes .................... 21 Fixed Stained Sections ........................ 21 Negative Controls .......................... 21 Sections Stained for Immunoglobulin G ...... 21 Sections Stained with DT-2 ................. 24 Frozen Stained Sections ....................... 24 Negative contrOlS C.COOOOOOCOOOOOOOOOOCOOOOO 24 Sections Stained for Immunoglobulin G ...... 24 Sections Stained with DT-2 ................. 24 DISCUSSION.OOOOOOOOOOOOOOOO....00...OOOOOOOOOOOOOOOOOOOO 31 SUT’IMARY 0.00.0000....0.0..O...0.00.0000...OOOOOOOOOOOOOO 33 iv APPENDIX 0....0...00...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO Lymphoma cases .................................... Analysis of Lymphoma Cases ........................ Fixed Stained Sections ........................ Negative Controls .......................... Positive Controls .......................... Sections Stained for Immunoglobulin G ...... Sections Stained with DT-2 ................. Frozen Stained Sections ....................... Negative Controls .......................... Positive Controls .......................... Sections Stained for Immunoglobulin G ...... Sections Stained with DT-Z ................. BIBLIOGRAPHY O...00......0......OOOOCOOOOOOOCOOOOCOOOOOO VITA 34 34 34 34 34 34 35 35 35 35 35 35 36 42 46 LIST OF TABLES Table Page 1 Buffer Formulas OOOOOOOOOOOOOOOOOOOOO0.0.0.0.0...0. 15 2 Immunoperoxidase Staining Procedure ............... 19 LIST OF FIGURES Figure Page 1 Negative control section of the medullary region in a fixed normal lymph node, lymph node 4. There is a general lack of staining of any lymphoid cells. Many plasma cells (solid arrows) are devoid of positive staining. Note macrOphage (Open arrow) contains cytOplasmic hemosiderin pigment. (lOOX) 0......0.0.0....OOOOOOOOOOOCOOOOOOOOOOOO 23 2 Medullary area of lymph node 4, section stained for IgG at dilution of 1:3000. Note plasma cell (arrow) with strong cytoplasmic staining. (128X) ............................. 23 3 Frozen lymph node number 5 cortex, stained for the presence of IgG with a dilution of 1:6000. Note the diffuse positive brown staining of the central follicular area and lack of staining in the mantle zone. (lOOX) .......... 25 4 Example of a frozen negative control section, lymph node number 5, cortex. (lOOX) .......... 27 5 Frozen lymph node section number 3, cortex, stained with DT-2. Abrupt margin at follicular-parafollicular junction (solid arrow). Note positive staining in the parafollicular area (solid arrow), and the many positive cells in the central follicular zone (Open arrow). (51X) .... .................27 6 Lymphoma ML-l, fixed negative control section. (128X) OOOOOOOOOOOOOOOIOOOOOOOIOOOOOOOOOOOOOOOO 38 7 Lymphoma ML-l stained for the presence of IgG with a dilution of 1:3000. Note the general lack of positive staining. (128X) ............ 38 vii Figure 10 Page Fixed lymphomatous lymph node number ML-2 stained for IgG, with a dilution of 1:3000. Note the general lack of staining throughout the section. (128X) .......................... 39 Fixed negative control section from lymphoma ML-3. (lOOX) OOOOOCCOOOCOOOOOCO0.000.000.0000. 41 Lymphoma ML-3 stained for IgG at dilution of 1:3000. Most cells contain a strongly pOSitive cytoplasm. (lOOX) OOOCOOOOOOOOOOOOOOOO 41 viii INTRODUCTION The differentiation of B and T lymphocytes is important in understanding the function, develOpment, and neoplasia of the immune system. Current methods of immunOperoxidase staining, in combination with specific antibodies, can distinguish B and T cells. These techniques, however, have not been adequately applied to canine lymphoid tissue. The objectives of this study were: 1. Determine if commercially prepared avidin—biotin complex (ABC) immunoperoxidase kits and antisera are of adequate specificity to be used in differentiating canine B and T lymphocytes in tissue sections. 2. Determine if DT-2 monoclonal antibody is useful in detecting T lymphocytes in tissue sections. LITERATURE REVIEW Embryology Lymph nodes contain several types of cells, among which are the B and T lymphocytes. During gestation, lymphoid stem cells are produced in the yolk sac membrane and migrate to the primary lymphoid organs, the thymus and avian bursa or its mammalian equivalent, (probably liver or bone marrow). There, they differentiate into B or T cells before migrating to the secondary lymphoid organs, the lymph nodes and spleen (Tizard, 1981; Valli gt al., 1981). Anatomy The normal lymph node is composed of a peripheral cortex, a central medulla and an indistinct region between these two areas termed the paracortex. Although there are minor species variations, such as a less distinct junction between the medullary and paracortical areas in man, and the apparent anatomic reversal of the cortex and medulla in pigs, these zones are functionally present in most mammals (Robb-Smith and Taylor, 1981). The cortex contains multiple cellular aggregates called follicles, of which there are primary and secondary types. The primary follicles are spherical collections of small lymphocytes located mainly in a peripheral portion of the cortex. In the face of immune stimulation, these may become secondary follicles (Robb-Smith and Taylor, 1981; Dellman and Brown, 1981). The secondary follicles are usually larger and contain a lighter staining germinal center. Here differing proportions of large and medium sized lymphocytes, as well as tingible body macrophages and dendritic reticular cells are located. A mantle zone surrounds the germinal center and is composed of a dense collar of small lymphocytes (Robb-Smith and Taylor, 1981; van den Tweel 33 $1., 1980). The paracortex is less cellular than the cortex, and occupies the space between the cortex and medulla. The unique feature of this area of the lymph node is the presence of small post-capillary venules. Tall endothelial cells line these venules which are believed to be capable of allowing large numbers of lymphocytes through their walls (Robb-Smith and Taylor, 1981; van den Tweel gt 31., 1980; Dellman and Brown, 1981) The medulla is composed of medullary cords and medullary sinuses. The medullary cords are branching and anastomosing projections of lymphoid tissue. They are intermeshed between the medullary sinuses, which are a network of vascular channels. The cell populations in the medullary cords vary with the reactive state of the lymph node. In antigenically stimulated states the numbers of plasma cells increase in direct proportion to the degree of stimulation (Robb-Smith and Taylor, 1981; Van den Tweel, 1980; Dellman and Brown, 1981) Immunologic Zones B—lymphocyte dependent zones are located within the follicles and the medullary cords. Follicular centers are the site of B-cell transformation. These transformed cells then migrate to the medullary cords where they mature into plasma cells . In this area the maturing plasma cells switch from synthesis of surface immunoglobulin to synthesis of secretory (cytoplasmic) immunoglobulin (Stein 2; al,, 1980). T lymphocytes are are found in highest concentrations in the paracortical zones and in lesser numbers in central follicular and parafollicular areas (Stein £5.2i-v 1980). The principle function of T lymphocytes is in modulating cell mediated immunity, such as graft rejection and delayed hypersensitivity (Parker, 1979; van den Tweel 22 31., 1980). Robb-Smith and Taylor, (1981) state that normal B and T lymphocytes are not readily distinguishable morphologically, and at present are only reliably differentiated by immunologic techniques. These techniques usually exploit the functional differences between these cells. When placed in a suspension of xenogeneic erythrocytes, T cells were observed to bind erythrocytes to the cell surface forming a "rosette" pattern (Tizard, 1982). Mitogen stimulation has been used to differentiate cells by observing the extent to which plant proteins, or lectins, bind to the surface of lymphocytes and cause them to divide. Some lectins bind only to B lymphocytes, others bind only to T lymphocytes and some bind to both (Tizard, 1982). These function tests can only be used with fresh tissue and cells in suspension, thus they have limited usefulness in differentiating T and B lymphocytes. Immunoglobulin appears to be the most useful marker for identification of the B-cell series, since all B lymphocytes possess cytOplasmic and/or membrane bound immunoglobulin (Parker, 1979; Seligmann, 1974; Harris gt 31., 1982; Lennert gt_gl., 1975). The development of human T-lymphocyte specific antisera has allowed a more accurate means of identifying this type of cell. In humans, antisera have been produced against a number of cell types, including thymocytes, peripheral T lymphocytes and T—series cells (Bruce £3 31., 1981; Martin gt 31., 1981; van de Gried 23 31., 1981; Warnke and Levy, 1980). In attempting to improve recognition of canine T cells, several antibodies have been developed (Gillman gt $1., 1980; Wulff $3.31., 1982a; Wulff 23 31., 1982b; McKenzie and Fabre, 1981). A recently develOped antibody is a murine monoclonal antibody identified as DT-2 (Wulff 25 31., 1982a). This antibody is a G2a subclass immunoglobulin developed against canine thymus cells. This marker identified puppy thymocytes, peripheral T cells, the majority of thoracic duct and bronchoalveolar lymphocytes. Immunostaining Methods The use of fluorescent antibodies to detect antigens has been in use since the early 1950s (Coons 23.2ir 1941; Coons and Kaplan, 1950). This method utilized fluorescein isothiocyanate (FITC), a yellow dye chemically bound to an antibody. The antibody-bound dye will then fluoresce a green light when viewed under an ultraviolet (UV) microscope. Fluorescent antibody techniques provide a means of identifying specific antigenic receptors or cell types in suspension or tissue sections. There are two immunofluorescent methods, the direct and indirect (Tizard, 1982). The direct immunofluorescent technique links the FITC dye to the primary antibody. This fluorescein tagged antibody, bound to its specific antigen, fluoresces when viewed under UV, visibly marking the detected antigen (Tizard, 1982). In the indirect technique an unconjugated primary antibody is initially bound to the specified antigen in the specimen. The antigen bound primary antibody is exposed to an FITC conjugated secondary antibody, specific for the primary antibody. Again when viewed under the UV microsc0pe visible evidence of the antigenic determinant is recorded (Tizard, 1982). Several disadvantages soon became apparent with the immunofluorescent procedure. The preparations were not permanent, specialized microscopy was needed and only fresh samples could be used. The development of enzyme labeled antibody techniques in 1966 overcame many of these shortcomings (Taylor, 1978; Nakane and Pierce, 1966). Basic techniques currently available to identify antigens in tissues include 1) direct immunOperoxidase 2) indirect immunoperoxidase 3) Peroxidase-antiperoxidase (PAP) and 4) the Avidin-Biotin Peroxidase Complex (ABC). The direct and indirect immunoperoxidase methods are analogous to the direct and indirect immunofluorescent techniques described previously. The only difference is in the marker that is bound to the conjugated antibody. In the peroxidase technique the FITC dye is replaced by a peroxidase enzyme molecule. This peroxidase enzyme reacts with a chromogen molecule (generally 3,3' Diaminobenzidine) to produce a brown product, which can be identified by routine light microscopy. The PAP method is estimated to be 10-1000 times more sensitive than indirect immunofluorescence (Taylor, 1980). The reasons for this in part are due to the number of labeling molecules (fluorescein or peroxidase) that bind to each secondary antibody molecule. The PAP complex binds three labeling molecules (Hsu gt al., 1981), while the indirect method binds only one or two. It is this greater sensitivity of the PAP method which allows its use in fixed tissue. This technique utilizes a primary antibody capable of binding to a specific antigen, and a secondary antibody which links the primary antibody to a peroxidase linked third antibody (peroxidase-antiperoxidase complex). Hsu gEDQI. (1981) recently develOped a three stage ABC technique which is based on the high affinity of biotin for avidin (Guesdon 23 31., 1979). This method utilized three reagents: 1) the primary antibody, specific for the antigen to be localized, 2) the secondary antibody, conjugated to biotin, which is capable of binding to the primary antibody and, 3) a complex of peroxidase conjugated biotin and avidin (Bourne, 1983). The ABC complex is a lattice containing many peroxidase molecules which forms an irreversible bond with the biotinylated antibody. This compares with the PAP method in which 3 molecules of peroxidase can be attached to each PAP complex. In addition, the ABC technique permits the use of antisera at far greater dilutions than the PAP method (Hsu gt'gl., 1981; Falini and Taylor, 1983). Hsu gt_gl., (1983) applied the ABC technique successfully to tonsils, lymph nodes, spleen and thymus. In this study, anti-B antibodies and anti-T antibodies stained these organs specifically for their respective B and T-cell dependent regions. The use of the ABC technique shortened the reaction time over the PAP technique and increased the staining intensity. In addition, the ABC method was more sensitive than the PAP technique. Using the ABC method IgG can be identified on plasma cells at a primary antibody dilution of 1:6400. The PAP technique generally requires primary antibody dilutions of 1:400 (Hsu 33 31., 1981). Procedural Considerations Several studies have been undertaken to determine the effects of various fixatives on surface and cytoplasmic immunoglobulin (Lewis 23.31., 1983; Banks $5.11., 1979; Giorno and Kohler, 1983; Brandtzaeg and Rognum, 1983; Pangalis g£_gl., 1981). These included 10% formalin, Zenker's fixative, B5, and Bouin's or Zamboni's fixing agents. All of these fixatives destroyed surface immunoglobulin. Cytoplasmic immunoglobulin was preserved with all fixatives studied. However, in tissues fixed with the above fixatives, immunoglobulin concentrations of at least 8 times higher were needed for detection (Brandtzaeg and Rognum, 1983). Giorno and Kohler (1983) reported that cytoplasmic lO immunoglobulin was masked by fixation with formalin, resulting in decreased sensitivity of immunoperoxidase staining. However, this immunoglobulin could be unmasked by pretreating the tissue sections with the enzyme trypsin. Trypsinization of tissue sections did not reverse the effects of the fixative on surface antigens. Various buffers have been used for diluents or rinsing in immunoperoxidase techniques. Many of these buffers contain sodium azide as a preservative which may be present in sufficient concentrations to prevent binding of peroxidase enzyme to its substrate and weaken staining. It has therefore been recommended that buffers containing sodium azide be avoided in immunOperoxidase procedures (Bourne, 1983; Pangalis gt.gl., 1983). Several factors have been reported to contribute to nonspecific background staining including antibodies of unwanted specificity (Taylor, 1979; DeLellis g£_gl., 1979). Taylor suggested that this may be countered by serial dilution of the primary antibody and selecting the concentration which gives the greatest contrast (Taylor, 1978). Monospecific antibodies may contain antibodies directed against two or more antigenic determinants in a molecule, leading to cross reactions. Taylor stated this could be circumvented by utilizing only highly purified monospecific antisera. He also stated that the difficulty in purification requires that each investigator be cautious 11 in the interpretation of results (Taylor, 1978). Bourne (1983) reported that collagen and connective tissue are highly charged tissues which attract protein including antibodies. She also stated that this can be avoided by adding an innocuous protein, e.g. nonimmune serum, to fill the charged sites. Endogenous peroxidase was normally found in some tissues, particularly those with red and white blood cells. Since endogenous peroxidase results in false staining, it is necessary to block or destroy it if tissues contain large numbers of these cells (i.e. lymph nodes). A solution of hydrogen peroxide was effective in removal prior to staining (Bourne, 1983). One researcher found that attempts to block endogenous peroxide with methanolic peroxide resulted in a decrease in the intensity of specific staining (Giorno and Kohler, 1983). Spurious staining may result from endogenous biotin. Biotin is a vitamin widely found in tissues, especially liver, adipose tissues, mammary gland and kidney. Wood examined lymph nodes and judged them to lack sufficient endogenous binding activity to interfere with the interpretation of avidin biotin staining (Wood 23 31., 1981). Research in Dogs Several researchers have attempted to classify both 12 normal and neoplastic lymphocytes in dogs. Dutta 23.31. (1977) classified B or T cells in suspension according to erythrocyte antibody complement (EAC) rosette formation and surface fluorescent immunoglobulin. However, other researchers have shown that the number of rosettes formed by canine thymus cells is variable and have suggested that this technique is unreliable in differentiating canine B and T lymphocytes (Beall §E_al., 1974). Holmberg (1976) attempted to differentiate canine B and T cells by mitogen response, surface immunoglobulin, rosetting and electron microscopy. Although these techniques provided some differentiation of lymphoid types it was the authors opinion that "more definitive techniques are needed to define B and T cells in the canine". MATERIALS AND METHODS Materials Immunologic Reagents Rabbit anti-canine immunoglobulin G (IgG) antiserum, specific for both heavy and light chains, was obtained commercially (a). The lyophilized antiserum was rehydrated with two milliliters of sterile distilled deionized water (DDH20), prior to use as suggested by the supplier. DT-2, a murine monoclonal antibody specific for canine T lymphocytes, was obtained from Fred Hutchinson Cancer Research Center and the University of Washington School of Medicine (b). Immunoperoxidase Kits Two ABC immunoperoxidase kits (c) were obtained commercially. One kit, specific for rabbit IgG, was used in detecting rabbit anti-canine IgG. A second kit, specific for all classes of murine IgG, was used in detecting murine antibody DT-Z. (a)Cappel Scientific Division, Cooper Biomedical Inc., Malvern, PA. (b)Seattle, WA. (c)Vectastain(R) ABC kit, Vector Lab, Burlingame , CA. 13 14 Other Reagents Several laboratory reagents including 3,3' Diaminobenzidine tetrahydrochloride (DAB), 30% hydrogen peroxide (H202), OCT compound, and isopentane were procured from several commercial sources (d). Phosphate Buffered Saline (PBS) and Tris-HCl buffers were prepared according to standard formulas (Table 1). Tissue Five canine lymph nodes — three mesenteric (LN-1, LN-2, LN—3), one prescapular (LN-4), and one popliteal (LN—5) — were obtained from normal dogs. These dogs were euthanized for reasons other than malignancy. Lymph nodes were removed from the dogs shortly after euthanasia. Methods Preparation g£_Tissue Specimens All lymph node specimens were hand—trimmed to remove adipose tissue and cut transversely at right angles to the long axis. These sections were cut approximately two millimeters thick, and included both cortex and medulla. Sections from each lymph node were either preserved in B5 fixative or by freezing in liquid nitrogen. (d)Sigma Chemical Co., St. Louis, MO. Lab-Tek Products, Harperville, IL. Fisher Scientific Co., Fair Lawn, NJ. 15 Table 1. Buffer Formulas Phosphate Buffered Saline 1. 8.0g NaCl 2. 0.2g KCl 3. 1.15g Na2HPO4 4. 0.2g KH2P04 5. DDH20 to make 1 liter 6. Add 1N. NaOH or 1N. HCl to adjust pH to 7.2 to 7.4. Tris HCl* 1. 25ml Tris 0.2 molar (24.23g tris[hydroxymethyl]aminomethane/l) 2. 44.7 ml. 0.1N HCl 3. DDH20 to make 100ml at pH 7.2 *Formula from Scientific Tables, 7TH Edition, (C)1970, published by Geigy pharmaceuticals. 16 Fixed tissue specimens were placed in B5 fixative for two hours. At the end of one hour these tissues were bisected into sections approximately one millimeter thick and returned to the B5 fixative. Two hours post fixation, these specimens were transfered to a 70% ethyl alcohol solution for transport and storage. Conventional methods were used to process and paraffin embed these specimens. After paraffin embedding, sections were cut at three microns and placed on standard glass microsc0pe slides. Frozen tissue specimens were prepared by placing individual specimens flat on cork discs (e). The entire section was covered by approximately one to two milliliters of OCT embedding media. This embedded tissue was snap frozen by immersion in cold isopentane, contained in a beaker held in liquid nitrogen. The specimen was frozen in approximately fifteen to thirty seconds. Frozen embedded specimens were then wrapped in aluminum foil, and stored at —56°centigrade (C) until sectioning. The frozen tissue was placed in the cryostat until the its temperature had equilibrated with that of the cryostat (-26°C). Specimens were then sectioned on the cryostat (f) (e)Lipshaw, Detroit, MI. (f)Model 50—AB Microtome, Lipshaw, Detroit, MI. 17 at six microns and placed on precooled -26°C microscope slides. Sections were adhered to the microsc0pe slides by warming the tissue with brief digital pressure applied to the underside of the slide, until the media had thawed. Slides were then held at -26°C prior to immunOperoxidase staining. Immungperoxidase Staining Procedure The immunoperoxidase procedure utilized in this project was carried out in the manner described by Hsu §E_aly (1981), and adapted for use in the ABC immunoperoxidase kit. All incubations were carried out at room temperature and both fixed and frozen tissue sections were maintained in a moistened state through the entire immunoperoxidase procedure. A moist chamber, consisting of a sealable plastic container lined by moistened paper hand towels, was used for all incubation steps. The slides containing the sections were laid flat on a rack made of plastic straws, which kept the slides off the bottom of the chamber. Neoprene 'O' rings(g), measuring approximately 1.5 centimeters in diameter, were placed so that the tissue sections were encircled. The reagents, used in the staining process, were deposited within the lumen of the 'O' ring, preventing the fluid from dipersing away from the tissue (g)Purchased at a local hardware store, manufacturer unknown. 18 section. Optimal concentrations of primary antibodies were established by serially diluting the antibodies in PBS. Dilutions used in this project were 1:2000 for the monoclonal antibody (DT—2) and 1:6000 for the rabbit anti—canine IgG antisera. Dilutions of 1:1000 and 1:3000 respectively were also used satisfactorily. Fixed tissue sections were deparaffinized and rehydrated by conventional histologic techniques utilizing xylene and a series of ethyl alcohol solutions in decreasing strengths (Table 2, step 1). Sections were then washed in DDH20 for five minutes, and the step by step procedure was followed as indicated in Table 2. Those sections fixed in B5 solution were processed to remove mercuric chloride crystals, as described by Luna (1968), by a standard established method. The sections then were counterstained with Erlich's hematoxylin for five minutes. The excess counterstain was removed by a final wash in tap water for five minutes, prior to dehydrating, clearing, and coverslipping. Dehydration and clearing were accomplished by reversing the rehydration and deparaffinization procedures. Frozen sections were fixed in ~26°C acetone before washing ten minutes in PBS. The step by step procedure outlined in Table 2 was followed. Sections were counterstained in Ehrlich's hematoxylin, for fifteen Table 2. IMMUNOPEROXIDASE B; Fixed Tissue 1. Deparaffinize and hydrate through xylene and graded alcohols series. 2. Rinse for 5 minutes in DDH20. 19 STAINING PROCEDURE Frozen Tissue 1. Fix tissue sections in -26°C acetone for five seconds. 2. Rinse for ten minutes PBS. All Specimens 3. Incubate the sections for thirty minutes in 0.3% hydrogen peroxide in methanol. 4. Wash in PBS for 20 minutes. 5. Incubate sections for 20 minutes with diluted normal serum which was prepared from species in which the secondary antibody is made. 6. Blot excess serum from sections. 7. Incubate sections for 30 minutes with primary antisera diluted in PBS. 8. Wash slides for 10 minutes in PBS. 9. Incubate sections for 30 minutes with diluted biotinylated antibody solution. 10. Wash slides for 10 minutes in PBS. 11. Incubate sections reagent. for 30 minutes with ABC 12. Wash slides for 10 minutes in PBS. 13. Incubate sections for 5 minutes in equal amounts of 0.02% H202 diluted in DDH20 and a 0.1% DAB solution diluted in 0.1 Molar Tris-HCl Buffer. 14. Wash sections for 5 minutes in tap water. Remove mercuric chloride crystals from B5 fixed tissue by conventional methods (Luna, 1968) 15. Counterstain, dehydrate, and mount. 20 seconds, and then washed in tap water. Dehydration, clearing, and coverslipping steps were similar to those used with fixed sections. Negative Controls Negative controls of fixed and frozen tissue were processed in the same manner as test slides except that PBS was substituted for the primary antisera. All other steps in the immunoperoxidase procedure remained the same. Negative controls were included in every staining effort. RESULTS Analysis gf Normal Lymph Nodes Lymph node sections, obtained from clinically normal dogs, were examined after immunostaining with the described ABC immunoperoxidase technique. These sections, from all lymph nodes (i.e. L1, L2, L3, L4, and L5), were described as a group according to the respective stain or control. Sections which stained positively for IgG, exhibited an intense red-brown coloration, usually confined to the cytOplasm of lymphoid cells. Sections which stained positively for DT-2 exhibited a black coloration of cells. Fixgg Stained Sections Negative controls: Cellular areas of the cortex and medulla stained negatively for IgG and DT-2 in negative control sections. Mild nonspecific staining was observed in collagen bundles and muscular tunics of large blood vessels. The cytoplasm of large mononuclear cells within the sinus network exhibited light brown staining characteristics. Sections staiggg Egg Immunoglobulin g: A few cells in central follicular areas and in mantle zones of the cortex, stained positively for IgG. These cells were medium sized lymphocytes with a nucleus of 10-15 microns and a moderate amount of cytoplasm. The medullary cords consistently had 21 22 Figure 1. Negative control section of the medullary region in a fixed normal lymph node, lymph node 4. There is a general lack of staining of any lymphoid cells. Many plasma cells (solid arrows) are devoid of positive staining. Note macrophage (open arrow) contains cytoplasmic hemosiderin pigment.(100X) Figure 2. Medullary area of lymph node 4 section stained for IgG at dilution of 1:3000. Note plasma cell (arrow) with strong cytoplasmic staining.(128X) Figure 2 24 the highest numbers of positively stained cells. These cells were usually present in small aggregates and were morphologically typical of plasma cells. Mild nonspecific staining of collagen was evident in the capsule and in media of muscular vessels. Sections stained yith.21;2: Negative staining occurred in all fixed tissue sections of lymph nodes stained with DT-2. Frozen Stained Sections Negative controls: Faint staining was observed in cells in central follicular areas of the cortex. Cellular areas of the medulla contained a few cells with a thin rim of cytoplasmic staining. Moderate nonspecific staining was observed in collagen bundles. Sections Stained £2; Immunoglobulig g: The intercellular spaces of the centers of secondary follicles in the cortex stained positively for IgG. Nonspecific background staining was observed in the outer cortex. Positive cellular staining was present in scattered cells throughout medullary cords. Moderate nonspecific staining of collagen bundles was also evident. Sections stained with DT-2: All sections stained with DT-2 exhibited moderate positive staining of cells surrounding secondary follicles. Many cells within larger 25 Note the lar stained 1cu (lOOX) f the central foll ining 0 Frozen lymph node number 5 cortex, Figure 3. for the presence of IgG with a dilution of 1:6000. area and lack of staining in the mantle zone. diffuse positive brown sta 26 Figure 4. Example of a frozen negative control section, lymph node number 5, cortex. (lOOX) Figure 5. Frozen lymph node section number 3, cortex, stained with DT-2. Abrupt margin at follicular- parafollicular junction (solid arrow). Note positive staining in the parafollicular area (solid arrow), and the many positive staining cells in the central follicular zone (open arrow).(51X) 27 Figure 5 28 germinal centers also stained positively with DT-2, while cells in the mantle zone of secondary follicles stained negative. Medullary cords stained indistinctly and the lymphoid cells in this area were negatively stained. A mild to moderate non—specific staining of collagen bundles was also evident. DISCUSSION This research has demonstrated that commercially prepared ABC kits, when combined with specific anti-canine antibodies, are effective in detecting antigens in frozen and fixed canine lymph node sections. When utilized with commercially obtained anti-canine IgG antisera, this procedure apprOpriately stained lymphocytes in areas corresponding anatomically to B-lymphocyte dependent areas, in fixed and frozen lymph node sections. When utilized with DT-2, a monoclonal antibody reported to be specific for canine T lymphocytes, positive staining of frozen lymph node sections was specific for areas described in man and animals to be rich in T lymphocytes. In the fixed sections stained for IgG, positive staining was readily evident in areas shown, in man, to contain B lymphocytes (Harris 33 31., 1982; Stein 9; _l., 1980). There was marked staining of many plasma cells. Negative staining of plasma cells may have occurred due to synthesis of other immunoglobulin classes (generally IgA, less often IgM and rarely IgD) (Harris 3; 31., 1982). Lack of staining in follicles may be related to a nonreactive status of the lymph node. In experiments done on animals, the number of cytoplasmic immunoglobulin 29 30 positive cells within the central follicular areas increased with the intensity and duration of antigenic stimulation (Stein 25 gl., 1980). Fixed lymph nodes, when stained with DT-2 were consistently negative. In man immunohistologic identification of T lymphocytes, utilizing fixed tissue, is also difficult. Anti-human T lymphocyte antibodies are generally directed against labile surface antigens, which are destroyed with fixation (Giorno and Kohler, 1983; Warnke and Levy, 1980). DT-2 may therefore be directed against a similar labile surface antigen. Evaluating the frozen sections stained for IgG was difficult due to sectioning artifacts and excessive background staining of collagen. As a result only approximately half of the sections were acceptable for interpretation. In these sections, an intercellular network of positive staining was observed in central follicular areas. A similar pattern has been reported in human lymph nodes stained for IgG (Stein 2; 31., 1980; Harris gt gl., 1982). This staining pattern was not observed in the control sections. Excessive background staining of the interfollicular zones and medullary cords, made it difficult to detect positively stained cells in these regions. Frozen lymph node sections stained with DT-2 stained consistently positive in known T-cell zones (Stein gg'gl., 1980; Falini and Taylor, 1983; Harris gg,gl., 1982). 31 These areas included the paracortical region and the area surrounding secondary follicles. Many cells in central areas of larger secondary follicles also stained with DT—2. Procedural factors can often affect the ABC immunoperoxidase staining procedure, making interpretation difficult. These factors include background staining, fixatives used, and spurious cellular staining. Background staining of collagen and connective tissue often hinders interpretation of ABC stained sections (Hsu gt‘gl., 1981). This was found to be a consistent problem in this study as well. The use of fixatives and buffers were evaluated prior to beginning the experiment. B5 fixative was chosen because it results in minimal loss of cytOplasmic antigens and maintains excellent nuclear morphology. Early attempts at staining often resulted in the poor staining of all slides. Various factors were altered including the incubation time with the ABC reagent. This was increased to sixty minutes, but still no improvement in staining occurred. A number of problems can cause lack of staining, one being drying out of tissue preparations during staining (Bourne, 1983). To correct this possibility, neoprene '0' rings were utilized. These helped to confine the reagents to the specimen area of the slides. This resulted in improvement so dramatic that sections were over stained. The incubation time of the ABC reagent was reduced 32 to thirty minutes with satisfactory results. Dilutions of antibody ranging from full strength, for the DT-Z, to 1:6000, for the anti-IgG, were evaluated in an attempt to increase specificity and decrease background staining. A dilution of 1:6000 of the anticanine IgG antisera was superior in the fixed lymph node sections, although in many cases 1:3000 was comparable. A dilution of 1:2000 produced the best results with DT-2 in frozen sections. The results from the frozen sections stained for IgG were capricious. Some stained adequately at 1:6000, while others were stained to excess. Dilutions of greater than 1:6000 could be evaluated to determine if further improvements result. The staining of frozen sections was particularly inconsistent in this study. Poor cryostat sections may have been a factor. In addition, it has been reported that in using the ABC on frozen tissues, increasing the incubation temperature and decreasing the time of incubation led to increased sensitivity and less background staining (Lewis g;,g;,, 1983). Further modifications of this type may improve the results in the frozen sections. SUMMARY Five normal canine lymph nodes and three lymphomatous lymph nodes were stained with a commercial avidin-biotin- peroxidase (ABC) kit, anti—canine immunoglobulin G and DT-2 (a monoclonal antibody reportedly specific for canine T lymphocytes) to determine the usefulness of these immunologic reagents in dogs. Both frozen and fixed tissue sections were utilized. The anti—canine immunoglobulin G had specific staining predominately in B cell areas, the medullary cords and the central follicular areas. The DT—2 antibody stained paracortical areas, interfollicular zones and the center of larger secondary follicles, all of which are known for T cell activity. No major modifications were necessary for adapting the ABC kit for use in canine lymphoid tissue. 33 APPENDIX APPENDIX Lymphoma Cases In addition to the five normal lymph nodes that were immunoperoxidase stained as described in the body of thesis, lymph nodes from three dogs with malignant lymphoma were also immunostained. The prescapular lymph nodes (ML-1, ML—2, and ML-3), were obtained from dogs that were presented by their oWners, to the Veterinary Clinical Center at Michigan State University. Analysis 2; Lymphoma Cases Fixed Stained Sections Negative contpgl Sections: Cellular areas throughout the lymphomatous lymph nodes stained negatively for IgG and DT—2 in control sections. The cortical and medullary areas in these sections could not be delineated. Mild nonspecific staining was observed in collagen bundles. In ML-3, many macrOphages contained a granular brown cytoplasmic pigment which stained positive for iron with the Prussian Blue stain. These cells were not observed in ML-l and ML-2. Positive control sections: Normal lymph node sections that were described in the results section of this thesis 34 35 were utilized as positive controls when interpreting the results of these lymphomatous lymph nodes. Sections Stained Egngmmunoglobulin g: ML-l sections contained rare randomly dispersed lymphomatous cells with moderately distinct cytoplasmic staining. ML-2 was negatively stained throughout the entire section. ML-3 had positive intense cytoplasmic staining in a majority of the cells throughout the section. Mild nonspecific staining of collagen bundles and media of muscular blood vessels was observed in these sections. Sections Stained yipp‘pgzgz The immunostaining of fixed sections of lymphomatous lymph nodes with DT-2 was not attempted. Frozen Stained Sections Negative Control Sections: Cellular areas in the lymphomatous lymph node sections stained negatively for IgG and DT-2. Mild nonspecific staining was observed in collagen bundles and muscular tunics of blood vessels. ML-3 had multifocal aggregates of macrOphages, which contained a cytOplasmic brown granular pigment. This pigment stained positively for iron with the Prussian Blue stain. Positive Control Sections: Normal lymph node sections that were described in the results section of this thesis were utilized as positive controls when interpreting the results of these lymphomatous lymph nodes. Sections Stained for Immunoglobulin 9; Frozen 36 sections of ML—l and ML—Z stained negatively for IgG. ML—3 had multifocal areas where numerous cells contained distinct positive cytOplasmic staining. All frozen sections had moderate nonspecific staining of collagen bundles throughout the sections. Sections Stained with DT-2: ML-l and ML—3 had an occasional positively stained cell randomly distributed throughout the sections. ML-2 stained negatively with DT-2. Mild nonspecific staining was observed in collagen bundles. 37 Figure 6. Lymphoma ML-l, negative control section. (128X) Figure 7. Lymphoma ML-l stained for the presence of IgG with a dilution of 1:3000. Note the general lack of positive staining. (128X) 38 Figure 6 Figure 7 39 Figure 8. Fixed lymphomatous lymph node ML—2 stained for IgG with a dilution of 1:3000. Note the general lack of staining throughout the section. (128X) 40 Figure 9. Fixed negative control section from lymphoma Figure 10. Lymphoma ML—3 stained for IgG at dilution of 1:3000. Most cells contain a strongly positive cytoplasm. (lOOX) 41 .1 r’ Figure 10 BI BLI OGRAPHY BIBLIOGRAPHY Banks PM: Diagnostic applications of an immunOperoxidase method in hematopathology. J Histochem Cytochem 27:1192-1194, 1979. Beall GN, Benfield JR, Kruger SR: Canine lymphocytes that form rosettes with human rbc. J Surg Res 17:330—337, 1974. Bourne J: Handbook 2; Immunoperoxidase Staining Methods. Immunochemistry Laboratory, Dako Corp., Santa Barbara, Cal., 1983. Brandtzaeg P, Rognum TO: Evaluation of tissue preparation methods and paired immunofluorescent staining for immunocytochemistry of lymphomas. Histochem J 15:655-689, 1983. 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Wood GS, Warnke R: Suppression of endogenous avidin-binding activity in tissue and its relevance to biotin-avidin detection systems. J Histochem Cytochem 29:1196-1204, 1981. Wulff JC, Deeg HJ, Storb R: A monoclonal antibody (DT-Z) recognizing canine T lymphocytes. Transplantation 33:616-620, 1982a. Wulff JC, DurkOpp N, Aprile J, gp'gl.: Two monoclonal antibodies (DLy-l and DLy-2) directed against canine lymphocytes. Exp Hematol 10:609-619, 1982b. VITA The author is a 1975 graduate of Michigan State University, College of Veterinary Medicine. He entered a residency program in Veterinary Pathology at M.S.U. in 1981 and completed his training in 1984. He fulfilled requirements for the Master of Science degree in Veterinary Pathology concurrently. 46