Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Microwave strategy for improving the simultaneous detection of estrogen receptor and galanin receptor mRNA in the rat hypothalamus.

In a attempt to improve the sensitivity of the simultaneous use of immunohistochemistry (IHC) with estrogen receptor (ER) and in situ hybridization (ISH) with a neuropeptide receptor, we first applied an existing microwave (MW) irradiation protocol for immunohistochemical detection of the estrogen receptor in frozen brain sections. Regions of interest were the preoptic area and the arcuate nucleus of the hypothalamus. ER signal was effective only after MW heating of sections in the two regions. Control sections without pretreatment exhibited no staining for ER. Second, the MW protocol was applied in a novel procedure that consists of evaluation of the expression of the galanin receptor mRNA with a radioactive riboprobe after MW pretreatment. The galanin receptor mRNA signal intensity obtained after heating was quantitatively at least as good or significantly increased according to the region, with no discernible loss of tissue morphology. Finally, we describe a novel application of MW pretreatment on the same frozen section processed with ER antibody and a radioactive galanin receptor riboprobe. The stainings for estrogen and galanin receptors were intense in many cells of the preoptic area, with very low background. These results show that both IHC and ISH can be significantly improved by subjecting frozen sections to MW heating before the double labeling. This approach may provide a potential method to answer the important question of whether or not estrogen has a direct action on the expression of a peptide receptor. (J Histochem Cytochem 49:901-910, 2001)     

Human specific anti-type IV collagen monoclonal antibodies,characterization and immunohistochemical application

Summary This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.     

Immunohistochemical demonstration of keratins in human ovarian neoplasms. A comparison of methods

A comparison of five immunohistochemical methods for the demonstration of keratins in human ovarian neoplasms using affinity-purified polyclonal rabbit antibody was made. The use of indirect immunofluorescence on frozen sections briefly fixed in acetone was found to be the most sensitive method and demonstrated keratin in all 14 primary and 1 metastatic ovarian epithelial neoplasms studied. Protein A-peroxidase, peroxidase--antiperoxidase (PAP), indirect peroxidase, or the avidin--biotinylated peroxidase complex (ABC) methods applied to formalin-fixed tissues were less sensitive and led to false negative results in 9 of 15, 1 of 15, 8 of 15, and 6 of 15 cases, respectively. A single case of dysgerminoma failed to reveal keratin by any method.     

Antigen Retrieval Using pH 3.5 Glycine-HCI Buffer or Urea Solution for Immunohistochemical Localization of Ki-67

A new antibody (MIB-1) has been described, permitting the demonstration of Ki-67 proliferation antigen in paraffin sections. However, satisfactory results were obtained only after subjecting tissue sections to microwave based antigen retrieval in citrate buffer solution. Other buffer solutions produce equivalent or better results and also permit use of the original Ki-67 antibody, which hitherto has been considered ineffective for paraffin sections.     

A new rapid immunohistochemical staining technique using the EnVision antibody complex.

Rapid immunohistochemical investigation, in addition to staining with hematoxylin and eosin, would be useful during intraoperative frozen section diagnosis in some cases. This study was undertaken to investigate whether the recently described EnVision system, a highly sensitive two-step immunohistochemical technique, could be modified for rapid immunostaining of frozen sections. Forty-five primary antibodies were tested on frozen sections from various different tissues. After fixation in acetone for 1 min and air-drying, the sections were incubated for 3 min each with the primary antibody, the EnVision complex (a large number of secondary antibodies and horseradish peroxidase coupled to a dextran backbone), and the chromogen (3,3'diaminobenzidine or 3-amino-9-ethylcarbazole). All reactions were carried out at 37C. Specific staining was seen with 38 antibodies (including HMB-45 and antibodies against keratin, vimentin, leukocyte common antigen, smooth muscle actin, synaptophysin, CD34, CD3, CD20, and prostate-specific antigen). A modification of the EnVision method allows the detection of a broad spectrum of antigens in frozen sections in less than 13 min. This method could be a useful new tool in frozen section diagnosis and research. (J Histochem Cytochem 49:623-630, 2001)     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

Analysis of proliferative activity in colorectal mucosa by immunohistochemical detection of proliferating cell nuclear antigen (PCNA)

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) has been suggested as a new approach for determinating proliferative activity in paraffin-embedded tissue. In a prospective study PCNA immunostaining was performed in 284 colorectal biopsies using monoclonal antibodies 19F4 (Ogata et al. 1987) and PC10 (Waseem and Lane 1990) and compared with the Ki67 method. From each site three biopsies were taken and a variety of fixation regimens for frozen and paraffin-embedded samples tested. For frozen biopsies methanol fixation at −20° C proved best. In paraffin sections PCNA could be detected after methacarn fixation as well as after controled fixation at 4° C in 4% paraformaldehyde for 1 h and in most biopsies routinely fixed with 10% formalin. However, the latter fixation regimens revealed additional PCNA-positive cells in the normal superficial colonic mucosal epithelium. Although the percentage of cells positive for PCNA was generally lower than for Ki67, the rates correlated in a highly significant fashion, both in frozen methanolfixed biopsies, and in paraformaldehyde-fixed paraffinembedded samples. PCNA immunohistochemistry revealed a similar proliferative activity in different parts of the large bowel. A higher proliferative activity was found in inflamed mucosa, adenomas, carcinomas and even in normal mucosa from patients with colorectal neoplasms. In routinely fixed biopies, the monoclonal antibody PC10 was superior to 19F4 because of considerably less background staining. However, in the routine material only a rough estimate of the proliferative activity was possible by PCNA immunohistochemistry using these antibodies, because unpredictable numbers of non-S-phase cells were also stained. Thus, it was concluded that reliable results are only obtainable after careful control of the fixation conditions. Taking this reservation into account, PCNA immunohistochemistry still represents a convenient method for measurements of proliferative activity in paraffin-embedded colorectal mucosa and can be applied using methanol-containing fixatives as well as after 4% paraformaldehyde fixation. Supported by a grant of the Werner and Klara Kreitz-Stiftung, Kiel to J.D.     

Enhanced Polymer One-Step Staining for Proliferating Cell Nuclear Antigen in Squamous Cell Carcinoma of the Tongue

A rapid enhanced polymer one-step staining for proliferating cell nuclear antigen [EPOS-PCNA) in formalin fixed, paraffin embedded sections of tongue squamous cell carcinoma is described. EPOS-PCNA was compared with PCNA immunohistochemistry using the avidin-biotin complex (ABC) method, with or without autoclave pretreatment. Significant correlation of PCNA labeling index (percentage of labeled cells/total cells counted) was observed between EPOS-PCNA and the immunohistochemical protocol using autoclave pretreatment. We consider this method a reliable, timesaving technique that would be useful in quantitative histopatbology and in performing double immunostaining.     

Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave-processed paraffin sections

Abstract. Antibodies against the proliferation-associated nuclear antigen (PCNA) and against the Ki-67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki-67 antibody had the inherent drawback in that it could only be used when fresh-frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin-fixed and paraffin-embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.
The aim of this study was to compare a recently prepared Ki-67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.
Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave-processing paraffin sections of formalin-fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave-treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki-67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.     

Analysis of proliferative activity in colorectal mucosa by immunohistochemical detection of proliferating cell nuclear antigen (PCNA). Methodological aspects and application to routine diagnostic material.

Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) has been suggested as a new approach for determining proliferative activity in paraffin-embedded tissue. In a prospective study PCNA immunostaining was performed in 284 colorectal biopsies using monoclonal antibodies 19F4 (Ogata et al. 1987) and PC10 (Waseem and Lane 1990) and compared with the Ki67 method. From each site three biopsies were taken and a variety of fixation regimens for frozen and paraffin-embedded samples tested. For frozen biopsies methanol fixation at -20 degrees C proved best. In paraffin sections PCNA could be detected after methacarn fixation as well as after controled fixation at 4 degrees C in 4% paraformaldehyde for 1 h and in most biopsies routinely fixed with 10% formalin. However, the latter fixation regimens revealed additional PCNA-positive cells in the normal superficial colonic mucosal epithelium. Although the percentage of cells positive for PCNA was generally lower than for Ki67, the rates correlated in a highly significant fashion, both in frozen methanol-fixed biopsies, and in paraformaldehyde-fixed paraffin-embedded samples. PCNA immunohistochemistry revealed a similar proliferative activity in different parts of the large bowel. A higher proliferative activity was found in inflamed mucosa, adenomas, carcinomas and even in normal mucosa from patients with colorectal neoplasms. In routinely fixed biopsies, the monoclonal antibody PC10 was superior to 19F4 because of considerably less background staining. However, in the routine material only a rough estimate of the proliferative activity was possible by PCNA immunohistochemistry using these antibodies, because unpredictable numbers of non-S-phase cells were also stained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of hormone receptors in breast carcinoma by immunocytochemistry and image analysis. II. Estrogen receptors

Frozen sections of 30 invasive breast carcinomas were stained for estrogen receptors (ERs) and the tumor cell proliferative rate by an immunoalkaline phosphatase technique. The stained sections were evaluated for ER by the microTICAS image analysis system. Seventeen tumors were ER positive and 13 were ER negative by image analysis. There was 93% concordance between the ER results obtained by image analysis and those obtained by biochemical methods. One case that was ER negative by image analysis was weakly positive by biochemical assay; a second case was ER positive by image analysis but ER negative by biochemical assay. Twelve of the 17 ER-positive tumors were diffusely positive while 5 displayed considerable intratumoral heterogeneity, with tumor cells exhibiting a broad range of intensity of receptor expression. In most cases, the image analysis ER status coincided with the progesterone receptor (PR) status, but in a large minority of cases (41%) the ER status and the PR status differed. Tumors with a high growth fraction (greater than 30%), as measured by Ki-67 immunostaining, were uniformly ER negative. The results of this investigation suggest that immunohistochemical staining of frozen sections for ER aided by automated image analysis (1) reliably detects the receptor in breast carcinoma, (2) allows for the assessment of heterogeneity within tumors and (3) may be used as part of a panel of antibodies to markers of potential prognostic importance in a single small tissue sample.     

Immunofluorescent Labelling of K-Papovavirus Antigens in Glycol Methacrylate Embedded Material: A Method for Studying Infected Cell Populations by Fluorescence Microscopy and Histological Staining of Adjacent Sections

Trypsin and protease V (pronase) were studied for their ability to enhance immuno-fluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 μm sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Immunofluorescent labelling of K-papovavirus antigens in glycol methacrylate embedded material: a method for studying infected cell populations by fluorescence microscopy and histological staining of adjacent sections

Trypsin and protease V (pronase) were studied for their ability to enhance immunofluorescent labelling of papovavirus antigens in glycol methacrylate embedded sections of organs infected with murine K-papovavirus. Treatment of Bouin's fixed sections with 0.4% trypsin for 30 minutes resulted in specific immunofluorescent staining equal to that seen in frozen sections and produced little if any loss of histological detail. Treatment with protease V resulted in less brilliant fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fluorescence and less satisfactory tissue preservation. Studies were then conducted to determine the fixative which would produce brightest specific fluorescent antibody staining of papovavirus-infected cells while providing clearest definition of intranuclear inclusions and best morphological detail in histologically stained adjacent sections. Brightest immunofluorescence staining was accomplished on material fixed in 96% ethanol/1% glacial acetic acid or Bouin's solution. These fixatives also gave clear definition of intranuclear inclusions with histological stains and provided excellent morphological detail. Phosphate buffered paraformaldehyde/picric acid and 3.7% formalin gave less satisfactory fluorescence and obscured intranuclear inclusions in histological preparations. Sections fixed in 4% paraformaldehyde, 4% paraformaldehyde/1% glutaraldehyde, and 0.5 M p-toluenesulfonic acid were negative for specific fluorescence. Glycol methacrylate, used with proper fixation and trypsin pretreatment of sections, provides a useful embedding medium for immunofluorescent identification of virus-infected cells, and the 1.0-2.0 micron sections routinely obtainable with GMA permit study of individual infected cells by fluorescent antibody and histological staining of adjacent sections.     

Comparative Study of Antigen Retrieval Heating Methods: Microwave, Microwave and Pressure Cooker, Autoclave, and Steamer

We present a study comparing the most popular beating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies; the microwave pressure cooker, extended microwave heating (5 min × 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min × 2 J. Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for “maximal retrieval” as a common end point for all laboratories.     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.     

Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections.

We have adapted existing microwave irradiation (MWI) protocols and applied them to the processing and immunoelectron microscopy of both plastic-embedded and frozen sections. Rat livers were fixed by rapid MW irradiation in a mild fixation solution. Fixed liver tissue was either cryosectioned or dehydrated and embedded in Spurr's, Unicryl, or LR White resin. Frozen sections and sections of acrylic-embedded tissue were immunolabeled in the MW oven with an anti-catalase antibody, followed by gold labeling. Controls were processed conventionally at room temperature (RT). The use of MWI greatly shortened the fixation, processing, and immunolabeling times without compromising the quality of ultrastructural preservation and the specificity of labeling. The higher immunogold labeling intensity was achieved after a 15-min incubation of primary antibody and gold markers under discontinued MWI at 37C. Quantification of the immunolabeling for catalase indicated a density increase of up to fourfold in the sections immunolabeled in the MW oven over that of samples immunolabeled at RT. These studies define the general conditions of fixation and immunolabeling for both acrylic resin-embedded material and frozen sections.     

Human follicular dendritic cells express CR1, CR2, and CR3 complement receptor antigens

The expression of complement receptors by human follicular dendritic cells (FDC) was investigated by immunohistochemical techniques by using polyclonal and monoclonal antibodies to antigenic determinants of CR1, CR2, and CR3. Upon optical immunohistochemical examination of frozen sections from human reactive lymph nodes and tonsils by a three-step immunoperoxidase technique, a strong staining of cell bodies and cytoplasmic extensions of FDC was observed in germinal centers with anti-CR1 and anti-CR2 antibodies. Staining for these antigens was also found on cytoplasmic extensions of FDC in the mantle zone and on the plasma membrane of B cells in the entire follicles. Staining of FDC with anti-CR2 antibody was more intense than that of B lymphocytes. Monoclonal antibodies directed against epitopes of the alpha-chain of CR3 weakly stained FDC in follicles in a similar pattern to that which was observed on adjacent sections with mouse monoclonal antibody KIM4 that only recognizes FDC in human lymph nodes. Immunoelectron-microscopy was performed on frozen sections of a lymph node involved with a centroblastic centrocytic B malignant lymphoma and a reactive tonsil with the use of rabbit F(ab')2 anti-CR1 antibodies and mouse monoclonal anti-CR2 antibody. All the plasma membrane of the cell body and cytoplasmic extensions of FDC in germinal centers and in the mantle zones homogeneously stained for CR1 and CR2 antigens. Fibroblastic reticulum cells were negative. The plasma membrane of tumoral B lymphocytes strongly stained with anti-CR1 and weakly stained with anti-CR2 antibodies. The presence of CR1, CR2, and CR3 on FDC is a unique surface characteristic of these cells that should optimally allow the cells to bind antigen/antibody complexes bearing any type of C3 fragment.     

Demonstration of cells of the mononuclear phagocyte lineage in the periodontium following experimental tooth movement in the rat

In this immunohistochemical study two monoclonal antibodies, ED1 and ED2, which recognize exclusively cells of the mononuclear phagocyte system (MPS) in the rat, were applied to study the presence of these cells during remodelling of the periodontal tissues following mechanically induced orthodontic tooth movement. The immunohistochemical procedure was carried out successfully on routinely processed, paraffin-embedded histological sections using the avidin-biotin-peroxidase-complex (ABC) technique. Cells of the MPS could be demonstrated on positive control sections of rat spleen and bone marrow. For the study of remodelling of the periodontal tissues only the ED1 antibody proved to be suitable. With this antibody, positive mononuclear and multinuclear cells, i.e. macrophages and osteoclasts, were seen throughout the periodontium even in the control animals. After the induction of orthodontic tooth movement activation of macrophages, osteoclasts and odontoclasts was demonstrable, all of them showing a clear-cut positive reaction to ED1.     

Mixed glycosidase pretreatment reduces nonspecific binding of antibodies to frozen tissue sections

Indirect immunohistochemical studies of frozen mouse tissues with mouse monoclonal antibodies yield, in general, suboptimal results primarily because of indiscriminate binding of secondary antibody to all mouse immunoglobulins, i.e., to the monoclonal reagent and to endogenous immunoglobulin nonspecifically trapped in the tissue. To reduce this nonspecific staining, frozen sections of mouse kidney were treated enzymatically. Optimal results were obtained following a 2 hr treatment with 20 mg/ml of mixed glycosidases (MG). This treatment reduced the nonspecific background staining of the interstitial spaces and blood vessels, but did not affect the reactivity of structurally bound immunoglobulin G (IgG) in the glomeruli or alter the reactivity of mouse renal tissue to the monoclonal antibody that recognizes an oligosaccharide antigenic determinant (SSEA-1). Eluates from enzyme-treated frozen tissue sections contained normally immunoreactive IgG in the form of dimers. These data indicate that MG treatment of frozen sections could be safely used to reduce the content of nonstructurally bound immunoglobulins in frozen tissues and thus improve the visualization of specific monoclonal antibody binding.