The comparison of methods to identify the presence of fibrocytes in formalin-fixed,paraffin-embedded archival cardiac tissue with coronary heart disease

The purpose of this study was to find the optimal technical approach to identify the presence of fibrocytes in formalin-fixed, paraffin-embedded archival cardiac tissue with CHD (coronary heart disease). Using the coexpression markers CD45 and αSMA, the presence of fibrocytes was examined by three different methods, including double immunohistochemistry staining, combination labeling of immunohistochemistry and immunofluorescence and double immunofluorescence labeling. Double immunohistochemistry staining was very difficult to identify the CD45⁺/αSMA⁺ fibrocytes. Although combination staining of immunohistochemistry and immunofluorescence has made it possible to evaluate the co-localization of CD45 and αSMA in the fibrocytes, this method was prone to produce many false positive cells. In contrast, CD45⁺/αSMA⁺ fibrocytes could be clearly recognized by double immunofluorescence labeling. In conclusion, double immunofluorescence labeling is the optimal technical approach to identify the presence of fibrocytes in routinely processed cardiac tissue with CHD.     

DEVELOPMENT OF AN OPTIMAL PROTOCOL FOR ANTIGEN RETRIEVAL: A ‘TEST BATTERY’ APPROACH EXEMPLIFIED WITH REFERENCE TO THE STAINING OF RETINOBLASTOMA PROTEIN (pRB) IN FORMALIN-FIXED PARAFFIN SECTIONS

The retinoblastoma (RB) gene, which encodes the nuclear RB protein (pRB), is believed to be involved in cell cycle control and cell differentiation. Studies have demonstrated that loss of RB function may play a role in tumour formation and progression of a variety of human tumours, such as bladder, lung, breast, and prostate cancers. The immunohistochemical detection of pRB expression in formalin–paraffin sections of human cancer has potential advantages of convenience, economy, and compatibility with routine surgical pathology practice. In practice, however, results using pRB antibodies on routinely processed, paraffin-embedded tissue have been inconsistent. In this study, the antigen retrieval (AR) method has been applied to the immunohistochemical detection of pRB in paraffin-embedded tissues and a ‘test battery’ approach has been developed to identify the principal variables that result in the optimal AR protocol. This approach includes the use of buffered solutions at pH 1, 6, and 10 with three different heating conditions (temperatures 120°C, 100°C, and 90°C). In the example described here with antibody RB-WL-1, the low pH solution with the microwave heating at 100°C proved most effective. Both fresh and routinely processed formalin–paraffin tissues of normal and bladder carcinoma were used for a comparison of the pRB immunostaining. The AR method was evaluated by comparing the immunohistochemical staining result on routinely processed formalin–paraffin sections with frozen sections of the same tumour. A consistent intensity of immunohistochemical staining for pRB was achieved using the identified optimal AR protocol on formalin–paraffin sections. All slides showed positive staining of pRB in normal mesenchymal and epithelial tissues. The pattern of pRB localization and intensity of staining was similar to that obtained in frozen sections, though the intensity obtained by AR treatment on paraffin sections was slightly to moderately stronger than that obtained in frozen sections. Once the protocol was identified, it was tested using routinely processed paraffin tissue sections of 245 cases of bladder carcinoma, with consistent pRB immunostaining results. The protocol described is simple to perform and gives reproducible results for evaluation of pRB expression by immunohistochemistry.     

Immunofluorescence staining of adenovirus in fixed tissues pretreated with trypsin.

We describe an immunofluorescence staining procedure for adenovirus in Formalin-fixed, paraffin-embedded tissue sections pretreated with a 0.25% trypsin solution. In trypsinized tissue sections stained with fluorescein-labeled antibody to adenovirus, viral antigen was brightly fluorescent and easily detected each time specimens were processed, and the nonspecific background fluorescence was always minimal. Viral antigen was nonfluorescent or only weakly fluorescent in tissue sections not pretreated with trypsin. Because pathology laboratories usually receive Formalin-fixed tissues for examination, this rapid and practical procedure can be routinely used to extend the diagnostic capability of conventional histopathology.     

Staining procedure to aid in assessment of bucrylate histotoxicity in tissue sections

Isobutyl-2-cyanoacrylate (bucrylate) is commonly used as a material for therapeutic embolization. Histologic sections of embolized tissue routinely stained with hematoxylin-eosin show the bucrylate as a translucent material. We outline a staining procedure that demonstrates bucrylate in tissue sections. It is useful in routinely prepared paraffin-embedded tissue, and it is also effective after embolized tissues have been processed with petroleum ether rather than xylene. This stain may be effectively utilized to assess harmful tissue reactions to bucrylate.     

Simultaneous detection of a cell surface antigen and apoptosis by microwave-sensitized TUNEL assay on paraffin sections

The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) technique has been described as a sensitive method for detection of apoptotic nuclei in tissues and preferential staining of apoptotic strand breaks. Short-term microwave pre-treatment, a non-enzymatic pre-treatment technique of antigen retrieval, has been demonstrated to optimize the TUNEL method for in situ detection of apoptotic cells in formalin-fixed paraffin-embedded tissue sections. In the present study, we sensitized internal mammary artery sections by short-term microwave pre-treatment and used a two-step indirect enzymatic method to gain as an end product differentially stained cells, namely TUNEL-positive cells and these positive for the surface marker von Willebrand factor (vWF). This technique enables to clearly distinguish between apoptotic, non-apoptotic and vWF-positive cells that are phenotypic for endothelial cells. Phenotypic identification of cells is simplified by double staining with cell surface markers. This rapid, sensitive and reproducible technique allows simultaneous detection of DNA fragmentation and phenotypic markers in the same paraffin-embedded human tissue section.     

Immunophenotyping of non-Hodgkin''s lymphomas using a panel of antibodies on paraffin-embedded tissues.

The use of monoclonal and polyclonal antibodies for the immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue has been limited by the fact that most antigens on lymphoid cells are denatured by histologic fixation, dehydration, and embedment. In this article the authors have analyzed a small panel of antibodies which represent exceptions to this rule, in that they identify denaturation-resistant determinants on leukocyte antigens in paraffin-embedded tissue. Monoclonal antibodies L26 [corrected] and 4KB5 label preferentially B cells, monoclonal antibody UCHL1 stains predominantly T cells, and monoclonal antibody MAC 387 reacts with granulocytes and some macrophages. A polyclonal antiserum raised against purified CD3 (T3) antigen, a T-cell-specific molecule, was also employed. This antibody panel was used to immunophenotype routinely processed tissue biopsy specimens from 61 non-Hodgkin's lymphomas (all of which had been previously phenotyped in cryostat sections). The lineage of the neoplastic cells was correctly identified in 32 of 34 (94%) cases of B-cell lymphoma, in 19 of 19 (100%) cases of T-cell neoplasm, and in 2 of 4 (50%) cases of histiocytic malignancy. It is concluded that this combination of antibodies is helpful in immunophenotyping non-Hodgkin's lymphomas when only paraffin-embedded tissue sections are available, although additional reagents of higher specificity are required to improve the identification of lymphomas.     

Diagnosis of human lymphoma with monoclonal antileukocyte antibodies

Two monoclonal antibodies have been produced that react with antigens present on human white cells. These reagents differ from other monoclonal antibodies of similar specificity in that the antigens they recognize are resistant to conventional tissue-fixation and embedding procedures. These reagents can therefore be used in immunocytochemical staining of paraffin-embedded tissue sections. We assessed the practical usefulness of this technique in the histopathological diagnosis of human lymphoid neoplasms by staining a wide range of routine surgical biopsy specimens of normal and neoplastic tissue (gathered from five institutions), using an indirect immunoperoxidase technique. In all 40 cases of non-Hodgkin's lymphoma, positive labeling of neoplastic cells was obtained with one or both antibodies. In contrast, no staining of neoplastic cells was observed in 60 samples of nonlymphoid neoplasms. We conclude that many of the difficulties encountered by histopathologists in distinguishing between lymphoid and nonlymphoid neoplasms may be overcome by immunohistologic labeling with monoclonal antibodies such as the ones we have studied.     

Methods in pathology. Immunophenotype of hairy cell leukemia in paraffin sections

The immunophenotype of hairy cell leukemia (HCL) was investigated using 20 routinely fixed paraffin-embedded tissue sections (12 bone marrows, six spleens, one liver, one lymph node) from 12 patients known to have this disease. A panel of antibodies was used, including anti-leukocyte common antigen (anti-LCA), B-lineage antibodies (LN2, MB2, L26), T-lineage reagents (MT1, UCHL1), monocytic (anti-cathepsin B) and myelomonocytic (anti-lysosyme, Mac 387) antibodies, and other less lineage-specific markers (anti-S-100, anti-alpha-1-antichymotrypsin (anti-alpha 1-ACT), anti-alpha 1-antitrypsin (anti-alpha 1-AT), anti-vimentin). Anti-LCA stained hairy cells in seven of the 12 bone marrows and consistently recognized hairy cells in the spleen, liver, and lymph nodes. Hairy cells generally reacted with B-lineage antibodies and were not labeled by T-lineage markers. No reactivity was noted with myelomonocytic antibodies, anti-S-100, anti-alpha 1-ACT, or anti-alpha 1-AT. Vimentin was expressed in the majority of cases. Tartrate-resistant acid phosphatase reactivity was demonstrated in three of the 20 routinely processed tissue sections. These data suggest that immunohistochemical studies of hairy cell leukemia in routinely processed tissue may be useful in diagnostic hematopathology and surgical pathology.     

Production of specific monoclonal antibodies to Aspergillus species and their use in immunohistochemical identification of aspergillosis

Two anti-Aspergillus murine monoclonal antibodies (MAbs), designated 164G and 611F, have been produced; both specifically recognize cytoplasmic antigens of A. fumigatus, A. flavus, and A. niger by enzyme-linked immunosorbent assay. The MAbs can identify Aspergillus spp. both in frozen sections by immunofluorescence and in paraffin-embedded clinical specimens by immunofluorescence and immunoperoxidase staining.     

Comparative studies on the detection of hepatitis B virus DNA in frozen and paraffin sections by the polymerase chain reaction.

The polymerase chain reaction (PCR) is an extremely sensitive technique that has been used for detection of DNA sequences in formalin-fixed, paraffin-embedded tissues. In order to verify that hepatitis B virus (HBV) DNA sequences are adequately preserved in routinely processed liver tissues, we performed PCR with five different primer pairs for HBV sequences on DNA extracted by two different methods from paraffin and frozen liver sections. The amount of PCR products obtained with DNA templates extracted by the proteinase K-SDS method from frozen sections was significantly larger than that from paraffin sections. However, boiling of deparaffinized sections in water containing Chelex-100 resulted in ample amounts of PCR products irrespective of the primers used. On Southern blots, the location of the bands of amplified DNA obtained by the different methods was consistent with the predicted size, suggesting that the viral sequences had not been altered by processing. Although freezing of fresh tissue yields quantitatively more HBV DNA, formalin fixation qualitatively preserves the viral DNA sequences adequately for detection by PCR. Therefore, formalin-fixed, paraffin-embedded tissues may be used for the detection of viral DNA sequences by PCR. Application of the described procedure to routinely processed tissues significantly broadens the applicability of this powerful diagnostic and investigative method.     

Comparison of in situ hybridization using different nonisotopic probes for detection of Epstein-Barr virus in nasopharyngeal carcinoma and immunohistochemical correlation with anti-latent membrane protein antibody.

BACKGROUND: The detection of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) may be of diagnostic importance, particularly in cases from nonendemic areas. For cellular localization of viral genomes, cold in situ hybridization methods for the demonstration of EBV-associated NPC remain difficult and relatively insensitive for routinely processed tissues. EXPERIMENTAL DESIGN: The aim of the present study was to assess the importance of tissue processing and the hybridization targets to improve the sensitivity of the cold in situ hybridization method. In situ hybridization was performed in six cases of NPC using three biotinylated EBV cDNA probes (BamHI W/IR1, BamHI Y/EBNA2, XhoI/latent membrane protein) and two cocktails of EBER and BHLF1 oligonucleotides labeled with fluorescein isothiocyanate on routinely fixed and paraffin embedded sections. In two cases, in situ hybridization was also performed on specially processed (ModAMeX) sections. Immunohistochemistry was used to detect EBV-induced antigens using monoclonal antibodies against latent membrane protein, EBNA2 and ZEBRA (BZLF1). RESULTS: All cases showed EBV nucleic acids regardless of the tissue preparation with the three cDNA probes and on routinely processed sections with EBER oligonucleotides. By using cDNA probes, the best EBV DNA signal was obtained with BamHI W without heating of slides in tissue sections processed by ModAMeX, which probably gives rise to large amounts of single stranded DNAs. All cases positive with cDNA probes were found to be positive with EBER oligonucleotides and negative with BHLF1. However, on routinely processed paraffin sections, the signals with EBER oligonucleotides were stronger than with BamHI W cDNA probe. Dual labeling with in situ hybridization and immunohistochemistry showed that the hybridization signals were restricted to malignant epithelial cells. Latent membrane protein expression was detectable in four of six EBV nucleic acid-positive cases on both ModAMeX and routinely processed sections. The anti-EBNA2 and anti-ZEBRA antibodies were found to be negative on the two cases processed by ModAMeX. CONCLUSIONS: Cold in situ hybridization, in particular with EBER oligonucleotides, appears to be more reliable than immunohistochemistry with anti-latent membrane protein antibody to detect EBV in NPC in routine pathology. These findings confirm a distinctive phenotype (latent membrane protein +/-, EBNA2-, ZEBRA-) of EBV-positive NPC. The negative staining for BHLF1 oligonucleotides further supports the viral latency.     

Double immunofluorescence labelling of routinely processed paraffin sections

Double immunoenzymatic labelling of routinely processed human tissues has been used in many histopathology laboratories to compare the expression patterns of pairs of antigenic markers. However, these techniques are time-consuming, prone to background staining, and rarely suitable for detecting two antigens present at the same site, since one label tends to obscure the other. This paper reports the use of immmunofluorescence for double labelling of pairs of molecular markers in routinely processed tissue. The primary antibodies are either monoclonal reagents of differing isotype/subclass, or antibodies from different species, and labelling is visualized on a conventional fluorescence microscope equipped with a cooled CCD camera. Images can be captured and adjusted using personal computer hardware and software. This approach could be used for a wide range of tissue markers and only minimal tissue autofluorescence was observed. The procedure is more rapid than enzyme-based techniques and avoids the problems of interpreting two antigens present at the same site. Its establishment involves relatively minor expenditure and it may represent the optimal technical approach to the co-localization of pairs of antigens in routinely processed tissue samples.     

Inhibition of endogenous peroxidase for the immunocytochemical demonstration of intermediate filament proteins (IFP)

The necessity for minimally fixed and processed cell and tissue preparations for immunocytochemical studies of sensitive antigens such as lymphocyte surface markers is well recognised. In order to avoid methanol and hydrogen peroxide, which have been shown to be deleterious for certain antigens, various compounds have been proposed for blocking endogenous peroxidase activity (EPA) in tissue preparations which are to be used in immunoperoxidase reactions. In the present study the deleterious effect of methanol/H2O2 on intermediate filament proteins was demonstrated in both frozen sections and paraffin-embedded tissue. The use of alternative reagents for the non-deleterious blocking of EPA is recommended for immunocytochemical staining with antibodies against intermediate filaments.     

Simultaneous detection of DNA fragmentation (apoptosis), cell proliferation (MIB-1), and phenotype markers in routinely processed tissue sections

 In situ DNA fragmentation assays have proved to be particularly useful in the detection of apoptosis in routinely processed, paraffin-embedded tissue sections. In the present study, a triple-antigen labelling technique was performed to demonstrate DNA fragmentation (apoptosis), cell proliferation (MIB-1), and phenotypic markers in the same tissue section. The in situ apoptosis assay was conducted with the TUNEL method developed by a avidin-biotin alkaline phosphatase complex (ABcomplex/AP). The proliferation-associated MIB-1 antigen was demonstrated in the second staining sequence by the avidin-biotin peroxidase method (ABC). The phenotypic markers chromogranin A or prostate-specific antigen (PSA) were visualized by the alkaline phosphatase anti-alkaline phosphatase method (APAAP) in the third staining sequence. The feasibility of this triple-labelling technique was tested in formalin-fixed, paraffin-embedded tissue of prostatic adenocarcinomas from 8 patients with recurrent, hormone-refractory disease. Although these tumours revealed marked neuroendocrine differentiation, cell proliferation and apoptosis were detected exclusively in non-endocrine (chromogranin A-negative) tumour cells that expressed PSA variably. The triple-labelling protocol described here allows the phenotypic characterization of proliferating and apoptotic cell populations in the same tissue section. It may be useful in studies of tissue kinetics in physiological and pathological processes. Received: 21 July 1998 / Accepted: 3 September 1998     

Comparative study for the detection of peritubular capillary C4d deposition in human renal allografts using different methodologies

Detection of peritubular capillary (PTC) C4d deposition in tissue sections of renal allograft biopsies became an important aid in the diagnosis of antibody-mediated rejection. Pathologists in many major transplant centers now routinely stain renal allograft biopsies for C4d. Currently, there are 3 commercially available antibodies. Two of these antibodies are monoclonal and are usually used with either a 3- or a 2-step indirect immunofluorescence (IF) methodology on frozen sections. A polyclonal antibody is used on formalin-fixed, paraffin-embedded tissue section with an immunoperoxidase detection system. The goal of our study was to compare these antibodies and methodologies in our renal allograft biopsy material. Twenty renal allograft biopsies with diffuse or focal PTC C4d staining, using immunofluorescence methods on frozen sections, were selected for this study. These biopsies were tested with the 3 commercially available anti-C4d antibodies (Biogenesis, Brentwood, Calif, cat no. 222-8004; Quidel Corporation, Santa Clara, Calif, cat no. A213; and ALPCO Diagnostic, Windham, NH, cat no. 004-BI-RC4D). Both monoclonal antibodies (Biogenesis and Quidel) were tested with a 3- and a 2-step indirect IF method on frozen sections. The polyclonal antibody (ALPCO) was applied to formalin-fixed paraffin sections using immunoperoxidase methodology. In selected cases, the polyclonal antibody was tested on frozen sections with a 3-step indirect IF method. To exclude possible false-negative staining with the IF method, we selected 10 additional biopsies that showed PTC margination of inflammatory cells, but were C4d-negative or only focally positive, and tested them with the ALPCO antibody on paraffin sections. We have found that all methodologies and antibodies tested provided adequate results with only minor differences between them. Perhaps the most sensitive method is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. We prefer the 2-step indirect IF method with the Quidel monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost. The advantages and disadvantages of the individual methodologies are discussed.     

Immunohistochemical staining for terminal deoxynucleotidyl transferase (TDT). An enhanced method in routinely processed formalin-fixed tissue sections

This report describes an improved technique for sensitive and specific localization of terminal deoxynucleotidyl transferase (TdT) in routinely processed paraffin-embedded, formalin-fixed tissue sections using DNAse pretreatment and the avidin-biotin complex (ABC) technique. This method is useful in identifying lymphoblastic lymphomas (14/15 cases positive), with all other B- and T-cell lymphomas tested negative for the reaction. Used in conjunction with monoclonal antibodies immunoreactive for T- and B-cells in paraffin sections this technique should prove helpful in immunophenotyping malignant lymphomas where fresh tissue is unavailable for study.     

Immunophenotypic analysis of acute lymphoblastic leukemia using routinely processed bone marrow specimens

Monoclonal antibodies have been recently developed that react with antigens expressed on T and B lymphocytes in routinely processed, paraffin-embedded lymphoid tissues. In this study, we assessed bone marrow clot and/or core biopsy sections of 19 cases of acute lymphoblastic leukemia (ALL) using routinely decalcified, B5- or formalin-fixed, paraffin-embedded sections and a panel of monoclonal antibodies, including LN1, LN2, L26, Leu-22, UCHL-1, and LCA. Each case had been previously phenotyped using freshly obtained aspirate material and a standard immunophenotypic protocol. Our results demonstrate the utility of the LN2 antibody in differentiating between precursor B-cell (pre-B) and precursor T-cell ALL. The LN2 antibody stained 11 of 12 cases of pre-B ALL and did not react with any of the seven T-cell ALLs. The other antibodies tested were less helpful. The Leu-22 antibody stained both pre-B and T-cell ALLs, while the results with UCHL-1 revealed peculiar nuclear staining of pre-B and T-cell ALLs; this we attributed to processing artifact. The L26 antibody reacted with only one case of pre-B ALL (also CD20 antigen positive), while the LN1 antibody did not react with any pre-B ALLs. Neither L26 nor LN1 stained any cases of T-cell ALL. The LCA antibody stained in only four (21%) of 19 cases, two pre-B and two T-cell ALLs. The results also suggest that this panel of antibodies may be useful in differentiating ALL from mature B-cell and T-cell lymphomas involving the bone marrow.     

Identification of histiocytic reticulum cells by the immunohistochemical demonstration of factor XIII (F-XIIIa) in human lymph nodes

Morphologically and enzyme histochemically distinguishable tissue macrophages and stromal cells of human reactive lymph nodes were characterized by the cytoplasmic presence of the subunit A of factor XIII and by the expression of surface antigenic determinants reacting with monoclonal antibodies directed against monocyte/macrophage populations (Mo 1, Leu M3) and HLA-DR antigens. The distribution of F-XIIIa positive cells was studied on formaldehyde-fixed paraffin-embedded sections with immunoperoxidase techniques. established on cryostat section with double immunofluorescence. Alpha-Naphthyl acetate esterase (ANAE) reaction was The immunophenotype was established on cryostat sections with double immunofluorescence. Alpha-Naphthyl acetate esterase (ANAE) reaction was carried out on these cryostat sections to identify tissue macrophages. The antibody against F-XIIIa detected histiocytes in both intra- and extra-sinusoidal locations which were ANAE+, Mo 1+, Leu M3+ and HLA-DR-. F-XIIIa was also present in fibroblast-like mesenchymal cells with the following phenotypic characteristics: ANAE-, Mo 1+, Leu M3+ and HLA-DR+. The anti F-XIIIa antibody did not stain lymphoid cells, granulocytes, epithelial cells, endothelial cells and mast cells. The immunohistochemical detection of F-XIIIa works on formaldehyde-fixed paraffin-embedded sections. The most promising application seems to be the identification of histiocytes in lymphoid and histiocytic proliferations.     

Ki-M6 immunostaining in routinely processed sections of reactive and neoplastic human lymphoid tissue

A monoclonal antibody, termed Ki-M6 (CD68), which shows a restricted reactivity to cells of the monocyte/macrophage system, has been evaluated primarily with the use of cryostat sections. In this study the authors could assess that the Ki-M6 antibody recognizes a fixation-resistant epitope in most human macrophages. The Ki-M6 immunoreactivity with monocyte/macrophage-related cells was established by testing on routinely processed samples of reactive and neoplastic lymphoid tissues; it was compared with the staining for vimentin (V9) and S-100 protein antibodies, with visualization of the stationary elements of lymphoid tissues, with the aim of establishing its value in the study of the nonlymphoid microenvironment. The Ki-M6 antibody reactivity could be achieved with Bouin-fixed, paraffin-embedded tissue sections, without any proteolytic treatment, with the use of the avidin-biotin complex (ABC) method, especially after overnight incubation time at 4 degrees C. Some reduction in antigenic reactivity was observed in B5- or formaldehyde-fixed samples. The antibody reacted with macrophages of all different lymph node compartments; a broad reactivity against cells of macrophage lineage, including multinucleated giant cells, was observed in epithelioid granulomas. Ki-M6-positive cells other than classic macrophages were the so-called "plasmacytoid T-cells" and cells displaying elongated cytoplasms with fibroblastic-like features. Granulocytes, follicular dendritic reticulum cells, and interdigitating reticulum cells did not reveal any reactivity with Ki-M6 antibody. In malignant lesions, neoplastic cells of follicular and diffuse B- and T-cell lymphomas, including large cell non-Hodgkin's lymphomas, and Reed-Sternberg cells of Hodgkin's disease were negative in all cases studied. This study shows that Ki-M6 seems to be another anti-macrophage-specific antibody that reacts, in routinely processed tissue sections, with tissue macrophages but not with accessory cells. Thus, it may be a valuable addition to vimentin and S-100 protein antibodies for investigation of the microenvironmental organization of lymphoid tissues both in normal and neoplastic conditions.     

Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase

An avidin-biotin enhanced immunoperoxidase procedure using monoclonal antibodies (AA1, AA3, and AA5) prepared against human mast cell tryptase resulted in intense staining of mast cells in paraffin-embedded tissue. The distribution of mast cells observed was similar to that seen when adjacent serial sections were stained using a standard procedure with toluidine blue, though the immunoperoxidase technique permitted the identification of significantly more mast cells. With monoclonal antibody AA1, immunostaining was entirely specific for mast cell granules, and there was negligible background staining in a range of tissues including lung, tonsil, colon, gastric mucosa, skin, and pituitary. There was no staining of antibody on basophils or on any other normal blood leukocyte. The technique was effective with tissue fixed in either Carnoy's or neutral buffered formalin, though the internal mast cell structure was better preserved with formaldehyde fixation. The immunoperoxidase staining procedure with monoclonal antibody AA1 is a highly specific and sensitive means for the detection of mast cells in routinely processed tissues.