Glycol methacrylate (GMA) embedding for light microscopy. II. Immunohistochemical analysis of semithin sections of undecalcified marrow cores.

A routine method allows bone marrow biopsy specimens to be embedded in glycol methacrylate (GMA), a water miscible plastic, and to benefit from the advantages of good morphology with immunoperoxidase detection of a wide range of cellular antigens useful in diagnosing and classifying various haematopoietic disorders. Marrow cores were fixed in cold Bouin's solution, rinsed in cold phosphate buffer, dehydrated in cold methanol, infiltrated and embedded in cold GMA, then polymerised at 4 degrees C. Sections were cut at 2 micron thickness with a Tungsten carbide knife in a Jung's high performance microtome (Autocut). Antigenecity was preserved when drying slides at room temperature but pronase digestion was necessary to re-expose the antigens in bone marrow biopsy sections embedded in GMA. Histostik, a new adhesive, was used to coat the glass slides to prevent section loss during enzyme digestion and immunostaining procedures. This method of adapting plastic embedding to undecalcified marrow cores preserves marrow architecture and cellular details and it can serve as a useful adjunct to analyse the bone marrow from patients with myeloproliferative and lymphoproliferative disorders. This technique may also be applicable in non-haematological malignant conditions which affect the marrow.     

How the fixation-embedding protocol affects the specificity and efficiency of immunocytochemical stains for gonadotropin subunits

This report describes a study designed to test factors that may affect the efficiency and specificity of stains for gonadotropins. These include chemical or freeze-fixation and dehydration, heat polymerization of the plastic embedding compound, dehydration in organic solvents, and etching. Specifically, postembedding stains for LH or FSH subunits were applied to 1-μm sections of (1) Araldite-embedded pituitaries that were either chemically fixed and dehydrated or freeze-fixed and freeze-dried; (2) Aldehyde-fixed pituitaries that were dehydrated in water-soluble glycol methacrylate (GMA) and embedded in GMA at 4°C; and (3) p-formaldehyde-fixed pituitaries that were embedded in paraffin. A fourth group of pituitaries was dispersed and grown in monolayers for 1–3 days. These were stained following glutaraldehyde fixation. The optimal dilution of the primary antisera varied with the protocol; however, the percentage of cells staining for beta subunits did not change. In contrast, postembedding stains showed that alpha subunit reactivity is masked or destroyed in pituitaries that are fixed in glutaraldehyde and embedded in Araldite. Alpha chain reactivity was detected (in 14% of cells) either after freeze-fixation and freeze-drying followed by Araldite embedding, or after 4% paraformaldehyde fixation and GMA embedding (in 17% of cells). Staining in paraffin-embedded pituitaries was seen in only 10% of the cells. Preembedding stains for alpha chains were strikingly sensitive, however, and immunoreactivity was seen in 18–26% of the population of monolayer cells. Thus, whereas the percentages of cells staining for beta subunits do not change following the use of most of the fixation and embedding protocols, alpha chain reactivity is destroyed by all but the mildest. These findings show that one can control or improve the specificity of the stains for LH and FSH by the fixation-embedding protocol.     

Differential antigen preservation during tissue autolysis.

Immediate fixation or snap freezing of tissue is ordinarily done to maximize antigen preservation for immunocytochemistry; however, delay in tissue allocation or spontaneous lymph node infarction can render tissue suboptimal for immunostaining. To test the effects of tissue autolysis/necrosis on the preservation of various lymphoid, epithelial, and mesenchymal markers, two lymph nodes (one with reactive lymphoid hyperplasia and one with metastatic ductal breast carcinoma) were evaluated for immunocytochemically demonstrated antigen preservation at 0-, 4-, 8-, 12-, 24-, 48-, and 72-hour intervals of autolysis at 37 degrees C. All specimens were stained by frozen section and formalin-fixed paraffin section immunocytochemical reactions with antibodies against CLA (CD45), UCHL-1 (CD45RO), L-26, kappa, lambda, anti-epithelial keratins (AE-1 and AE-3), epithelial membrane antigen, and vimentin. Frozen sections were additionally stained for Leu-1 (CD5), Leu-2a (CD8), Leu-3a+b (CD4), Leu-4 (CD3), and Leu-14 (CD22). The most resilient lymphoid antigen preservation was observed with CLA and UCHL-1, both exhibiting immunoreactivity at 72 hours in both frozen and fixed preparations. L-26 showed similar reactivity in frozen sections, but detectable antigen was observed only up to 24 hours in formalin-fixed tissue. Leu-2a proved to be the most labile antigen, persisting for only 12 hours in frozen sections. The epithelial markers epithelial membrane antigen and AE-1 exhibited excellent antigenic preservation in both frozen and fixed preparations; AE-3 persisted well in frozen section but was not demonstrated in fixed tissue. Vimentin immunoreactivity was vastly superior in frozen, as compared with fixed, tissue sections. Most antigens showed remarkable preservation despite morphologic degradation; however, differential antigenic resilience was demonstrated. Knowledge of this variation in antigen decay is critical for evaluation of immunoperoxidase phenotypic studies of autolyzed or necrotic tissue.     

Immunohistochemical detection of c-erbB-2 protein in human benign and neoplastic prostate.

Both the polyclonal anti-c-erbB-2 peptide antiserum pAB 60 and the monoclonal anti-c-erbB-2 protein antibody mAB-1 detect the c-erbB-2 protein in human breast adenocarcinomas. We investigated c-erbB-2 expression in adult human benign hyperplastic and neoplastic prostates, using the avidin-biotin complex immunoperoxidase method. Formalin-fixed, paraffin-embedded specimens of benign hyperplastic prostate (13), prostatic adenocarcinoma (22), and prostatic adenocarcinoma lymph node metastases (two) were tested with pAB 60. Ten formalin-fixed, paraffin-embedded specimens of prostate adenocarcinoma, 11 frozen sections of benign hyperplastic specimens, and eight frozen sections of prostate adenocarcinoma were tested with mAB-1. Our results demonstrated consistent detection of c-erbB-2 immunohistochemically in frozen sections of both benign and malignant prostate. Preincubation of pAB 60 with the immunizing peptide blocked subsequent reactivity with prostatic tumor tissue, indicating specificity. However, fixation and processing protocols significantly affected the reactivity of the antigenic determinants detected by these antibodies, as mAB-1 was nonreactive with formalin-fixed, paraffin-embedded prostatic tissues. Differential reactivity of pAB 60 with malignant rather than benign glands was maximized by exposure of the specimen to the antibody at 4 degrees C rather than 22 degrees C. The most frequently observed staining pattern with both antibodies was cytoplasmic. However, mAB-1 produced distinctly membranous staining in two frozen specimens of benign hyperplasia and one specimen of prostate cancer.     

Axillary sentinel lymph node examination in breast carcinoma.

OBJECTIVE: To evaluate whether the type of pathologic examination of breast sentinel nodes (frozen section, step sections, and immunoperoxidase staining) results in different percentages of nodes positive for metastatic disease. DESIGN: Twenty-eight consecutive patients with breast sentinel node biopsies were evaluated by step-sectioning the sentinel node(s) along with performing immunoperoxidase stains for low-molecular-weight cytokeratin and epithelial membrane antigen. SETTING AND PARTICIPANTS: The patients were from a university hospital and large private hospital. MAIN OUTCOME MEASURES: The results of the step sections and immunoperoxidase stains were compared with routine examination, that is, intraoperative frozen section along with a single hematoxylin-eosin slide. RESULTS: Nine cases were positive by routine evaluation, 10 by step sections, and 11 by immunoperoxidase staining. CONCLUSIONS: The large, multi-institutional studies of sentinel node utility must take into account the surgical pathology methods used to evaluate these specimens so that uniform techniques, which reliably predict the status of the axillary nodes, can be instituted at all institutions that use this procedure.     

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues.

Routine fixation and paraffin embedding destroys many hematopoietic and lymphoid differentiation antigens detected by flow cytometry or frozen section immunohistochemistry. On the other hand, morphologic evaluation is difficult in flow cytometric or frozen section studies. A simplified three-step plastic embedding system using acetone-fixed tissues embedded in glycol-methacrylate (GMA) resin has been found to provide both excellent morphologic and antigenic preservation. With our system, a wide variety of antigens are detected in plastic sections without trypsinization or prolonged embedding procedures; pan-B (CD19, CD22), pan-T (CD7, CD5, CD3, CD2), T-subset (CD4, CD8, CD1, CD25) markers as well as surface immunoglobulin and markers for myeloid and mononuclear-phagocyte cells are preserved. In summary, modifications of plastic embedding techniques used in this study simplify the procedure, apparently achieve excellent antigenic preservation, and facilitate evaluation of morphologic details in relation to immunocytochemical markers.     

Immunoperoxidase staining for identification of Aspergillus species in routinely processed tissue sections.

AIMS: To evaluate the performance of an immunoperoxidase stain using the monoclonal antibody EB-A1 to detect Aspergillus species in formalin fixed, paraffin wax embedded tissue. METHODS: The monoclonal antibody EB-A1 directed against galactomannan was used to detect Aspergillus species in 23 patients with suspected or confirmed invasive aspergillosis. Immunostaining was performed on formalin fixed, paraffin wax embedded tissue using the streptavidin-biotin method and compared with conventional haematoxylin and eosin, periodic acid-Schiff, and Gomori-Grocott stains. Results of immunostaining were semiquantitatively analysed with regard to both intensity of staining and number of positively staining micro-organisms. Tissue sections from 16 patients with confirmed invasive mycoses due to Candida species, Apophysomyces elegans, Rhizopus oryzae, Pseudallescheria boydii and Histoplasma capsulatum were used as controls. RESULTS: In 19 (83%) of 23 cases invasive aspergillosis was confirmed by both histological examination and culture (18 Aspergillus fumigatus and one A flavus). Immunoperoxidase stains were positive in 17 (89%) of 19 cases including one case of disseminated infection due to A flavus. Furthermore, the immunoperoxidase stain was positive in a culture negative tissue section with histological evidence of mycelial development, indicating the presence of Aspergillus species. Some cross-reactivity was observed with the highly related fungus P boydii, although the number of mycelial elements that stained was low. CONCLUSIONS: Immunoperoxidase staining using the monoclonal antibody EB-A1 performs well on routinely processed tissue sections and permits detection and generic identification of Aspergillus species, although it was no better than conventional histopathology in identifying the presence of an infection. An additional advantage is that the immunostain may help to provide an aetiological diagnosis when cultures remain negative.     

Subject Index

Abstract

Acrylic resin mixtures are now widely used as embedding media for the preparation of tissue sections. Most of these mixtures are based on 2-hydroxyethyl methacrylate (glycol methacrylate, GMA). Resin embedding preserves tissue components far better than paraffin, celloidin or frozen sections. The present review describes the basic principles and trouble shooting, in particular: the chemical and physical properties of GMA, and components used for GMA mixtures; fixation of tissues for resin embedding; methods for dehydration; microtomy; stretching on water and mounting in relation to the final dimensions of GMA sections; staining of GMA 3embedded tissue sections; and the use of GMA resins in immunohistochemistry. In addition, standard, step by step procedures for embedding tissues in GMA is included. (The J Histotechnol 19:297–311, 1996)     

Expression of the antigen detected by the monoclonal antibody Ca 19.9 in human breast tissues

Summary The incidence and significance of the expression of the antigen defined by the monoclonal antibody Ca 19.9 (Sialyl Le^a) has been assessed in human breast tissue. Frozen and formalin-fixed, paraffin embedded specimens of normal, hyperplastic, pregnant breast and carcinomas were examined using an immunoperoxidase technique.Ductal and acinar epithelium of normal and hyperplastic tissues showed variable reactivity in frozen sections but there was a reduction in staining in comparable samples after fixation and processing, such that in many instances only focal ductal epithelium reacted. A distinctive feature in the pregnant breast was the absence of staining in acini showing differentiated secretory activity, despite a reaction in adjacent non-secretory acini and ducts.The overall incidence of detection of the Ca 19.9 antigen in breast carcinomas was 62%, but in half of these only a small number of cells stained. A significant relationship between expression of Sialyl Le^a and poor differentiation of carcinomas was identified, but there was no correlation with local lymph node status. In contrast to the non-malignant tissue fixation and processing had little effect on the reactivity of carcinomas. It is suggested that this difference may be quantitative in nature, with malignant breast showing much greater expression, or be related to organisation of the antigen.The observations concerning carcinomas and pregnant breast indicate that the synthesis of the Ca 19.9 antigen is related to the state of differentiation and functional activity of human breast.     

Demonstration of hepatitis B-surface antigen in liver biopsies. A comparative investigation of immunoperoxidase and orcein staining on identical sections of formalin fixed, paraffin embedded tissue.

Staining of 1000 consecutive liver biopsies with orcein showed positive reaction in the cytoplasm of ground-glass hepatocytes in 18 of the biopsies. On new sections of the 18 orcein positive biopsies immunoperoxidase staining was performed in order to demonstrate hepatitis B-surface antigen (HBsAg). After destaining the same sections were stained with orcein. All biopsies showing positive orcein staining showed positive immunoperoxidase reaction. The number of positive cells was in biopsies with less than 20 positive cells per mm2 biopsy larger using the immumoperoxidase staining than with orcein staining. Further the staining contrast was more pronounced. Immunoperoxidase staining thus seems more sensitive than orcein staining for demonstrating HBsAg in liver tissues. Orcein stains HBsAg in liver tissue even though the antigen determinants are blocked by antibodies.     

Methods in laboratory investigation. Immunoelectron microscopic methods for demonstration of antigens on normal human melanocytes and other epidermal cells

Different procedures for fixation and processing were evaluated in order to examine the antigenic profile of melanocytes and other epidermal cells for immunoelectron microscopy. For this purpose the monoclonal antibodies anti-HLA-A, B, C, anti-HLA-DR, anti-T6, and the melanoma-associated monoclonal antibody NKI /C-3 were used as markers. Fixation with periodate-lysine-paraformaldehyde yielded better antigenic and ultrastructural preservation than 3% paraformaldehyde or picric acid-paraformaldehyde did. Skin was further processed by five different methods: (a) 15-micron frozen sections; (b) 75-micron agar-embedded, tissue chopper sections; (c) 15-micron polyethylene glycol-embedded sections; (d) epidermal cells in suspension; and (e) epoxy sections (postembedding staining) were prepared for the immunoperoxidase procedure. Antigenicity was best preserved in the cell suspension method and somewhat less, but with a similar staining distribution, with the first three methods. Staining with the polyethylene glycol-embedded sections was only achieved if they were left free-floating in buffer; no staining was observed when the sections were mounted on glass slides and left to dry overnight at 37 degrees C. Epidermal cells remained unreactive in the postembedding method, even after etching. Ultrastructural preservation of the agar-embedded sections and the cells in suspension was superior to the other preembedding methods. Melanocytes mostly showed moderate staining for HLA-A, B, C and slight staining for the antigen that is recognized by NKI /C-3. The latter was further demonstrated on Langerhans cells and indeterminate cells which also expressed HLA-A, B, C, T6, and HLA-DR antigens.     

How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only approximately 30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.     

The application of immunoperoxidase methodology for the visualization of prolactin binding sites in human prostate tissue.

In previous studies the immunoperoxidase method was used to detect intracellular prolactin binding sites in epithelial cells of normal and neoplastic rat prostate. As an extension of this work, the same approach was used to test for and to localize prolactin binding sites in autopsy and biopsy specimens of formalin fixed, paraffin embedded human prostate. Rehydrated tissue sections were exposed to varying concentrations of human placental lactogen or human prolactin and then to human placental lactogen or human prolactin antisera. The loci of hormone binding were visualized by an immunoperoxidase staining sequence. Hormone pretreatment produced immunospecific, dose related staining inside epithelial cells of normal, hyperplastic, and neoplastic human prostate indicative of intracellular prolactin binding. The patterns of intracellular hormone binding in human prostate were similar to those seen previously in rat prostate tissue. The possible involvement of prolactin in prostatic cancer and the potential diagnostic value of the immunoperoxidase approach to hormone binding are discussed.     

Frequent positive staining with NKI/C3 in normal and neoplastic tissues limits its usefulness in the diagnosis of cellular neurothekeoma

NKI/C3 originally was described as a marker for melanoma. Recently, it resurfaced as a marker to separate cellular neurothekeoma from other dermal tumors in the differential diagnosis. To determine the sensitivity and specificity of NKI/C3, we evaluated its staining pattern in 709 normal and neoplastic tissues, including 92 dermal tumors, using tissue microarrays and conventional sections. We found that although NKI/C3 is positive in only a few normal tissues, it stains a broad spectrum of neoplastic tissues. NKI/C3 is also positive in many dermal tumors of possible histiocytic origin, including juvenile xanthogranuloma (6/10), atypical fibroxanthoma (4/12), cellular fibrous histiocytoma (5/10), reticulohistiocytoma (3/6), and xanthoma (8/10). However, it is negative in epithelioid cell histiocytomas (0/7) and Langerhans cell histiocytosis (0/9). Given the wide spectrum of positive staining in morphologic mimics of cellular neurothekeomas, pathologists should be cautious when making this diagnosis based solely on positive staining with NKI/C3.     

Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation.

AIMS--To investigate the effects of fixation on the immunohistochemical demonstration of c-erbB-2 oncoprotein using paraffin wax and cryostat sections; to compare c-erbB-2 expression in non-neoplastic and neoplastic gastric tissues. METHODS--Adjacent blocks of tumour and non-neoplastic tissue from four gastrectomy specimens were put into a panel of 10 fixatives including acetone, B5, Bouin's fluid, Carnoy's fluid, buffered formalin, formol dichromate, zinc formalin, 4% paraformaldehyde, periodate-lysine-paraformaldehyde (PLP) and periodate-lysine-paraformaldehyde-dichromate (PLPD) before embedding in paraffin wax for sectioning. Similar tissue blocks were snap frozen and cryostat sections were postfixed in these fixatives, either alone or in combination, before immunostaining. RESULTS--In paraffin wax embedded sections the best fixative was PLP, and in frozen tissues the best results were obtained after fixation of cryostat sections in buffered formalin followed by cold methanol and acetone. Applying these fixatives to samples from a further 16 gastrectomy specimens, strong membrane staining of c-erbB-2 protein was found in the tumour in eight of 16 cases (50%) using paraffin wax sections, and staining was stronger in the better differentiated carcinomas. For frozen tissues, positive membrane staining was found in all gastric adenocarcinomas, but differential staining intensity associated with tumour differentiation could not be detected. CONCLUSIONS--These results indicate that fixation and paraffin wax embedding affect the results of immunohistochemical demonstration of c-erbB-2 in gastric cancer. The choice of fixative is critical in the demonstration and evaluation of c-erbB-2 protein expression by immunohistochemistry in gastric carcinomas. Staining results also vary depending on whether frozen or paraffin wax embedded tissues are studied.     

An evaluation of potentially suitable fixatives for immunoperoxidase staining of estrogen receptors in imprints and frozen sections of breast carcinoma

The estrogen receptor (ER) content of breast carcinoma is generally accepted as valuable in predicting clinical outcome and tumour response to hormonal manipulation. We applied a new immunocytochemical assay for estrogen receptors (Abbott ERICA Monoclonal) to 20 breast tumours, and examined the efficacy of 16 fixation procedures before immunoperoxidase staining of frozen sections and imprint preparations. Our findings indicate that the fixatives of choice are periodatelysine-paraformaldehyde at 22 degrees C, or 10% formalin followed by acetone at -10 degrees C. These fixation procedures are simpler, less time-consuming, and provide superior staining, tumour cytomorphology and higher ER values than the 3-reagent sequence recommended by Abbott Laboratories. There was a significant correlation between the ER scores in the frozen sections and the imprints. Positive ER cytosol results correlated with the staining index in the frozen sections, and the ER scores in the imprints. We conclude that imprints are suitable preparations for ER analysis by the immunoperoxidase technique, particularly for small tumour specimens.     

A novel method for optimum biopsy specimen preservation for histochemical and immunohistochemical analysis

A novel method has been developed for optimally processing biopsy specimens combining freeze-substitution with low-temperature plastic embedding. Immunohistochemistry and conventional histochemical stains were all readily performed on tissue displaying high-quality morphologic preservation. Labile antigens, especially lymphoid cell surface antigens, were well preserved. This new method avoids the need for tissue fixation and combines the superior morphologic preservation of fixed embedded tissue with the reactivity of cryostat sections. This method ensures that diagnostic information from even the smallest biopsy specimen is maximized because a wide range of phenotypic markers can be applied and evaluated in relation to high-quality morphologic preservation of tissue. Biopsy specimens are stored at room temperature without loss of tissue-specific characteristics during storage.     

Detection of Helicobacter pylori in gastric biopsy and resection specimens.

AIMS: To compare the sensitivity of detecting Helicobacter pylori in gastric biopsy and resection specimens using tinctorial and silver impregnation stains, immunohistochemistry and the polymerase chain reaction (PCR). METHODS: Formalin fixed, paraffin wax embedded tissue from 33 gastric biopsy specimens (26 showing chronic gastritis and seven showing low grade mucosa associated lymphoid tissue (MALT) lymphoma) together with blocks of uninvolved mucosa from gastrectomy specimens for MALT lymphoma (five cases) were studied. Consecutive sections were stained using haematoxylin and eosin, Giemsa, the Warthin-Starry silver stain, and a polyclonal antibody directed against H pylori using an immunoperoxidase technique following heat induced antigen retrieval. PCR analysis of DNA extracted from a further section was carried out using primers which amplified a 411 base pair fragment of the urease A gene. RESULTS: H pylori was detected in 14 (37%) sections stained with haematoxylin and eosin, 21 (55%) with Giemsa, 23 (61%) with Warthin-Starry, and 25 (66%) stained with the antibody. Seventeen (45%) cases were positive on PCR. Immunohistochemistry was positive in all cases in which H pylori was detected by other methods. CONCLUSION: Immunohistochemistry using an immunoperoxidase technique following heat induced antigen retrieval for detecting H pylori in gastric biopsy and resection specimens is highly sensitive and easy to use.     

The effect of fixation and trypsinization on the immunohistochemical demonstration of intracellular immunoglobulin in paraffin embedded material

Using an indirect labelled immunoperoxidase technique the influence of fixation time on the antigenicity of intracellular immunoglobulin in lymphoid tissue fixed in buffered formalin has been investigated. Within a fixation period of 96 hours a decrease of 15% of stainable immunoglobulin containing cells was found, for every 24 hours the fixation time was prolonged. By comparing sections from tissue fixed in buffered formalin and selected fixatives (Lillie's AAF, Bouin's fluid, Clarke's fluid and 96% ethanol 1% acetic acid (E--A) processed at 4 degrees C and at 25 degrees C) an increased number of stained immunoglobulin containing cells was found in tissue fixed in Lillie's AAF, Bouin's fluid, Clarke's fluid and E--A processed at 4 degrees C. No difference was found between tissue fixed in buffered formalin and E--A processed at 25 degrees C. In addition the effect of pretreatment of the sections with trypsin on the number of stainable immunoglobulin containing cells was investigated. Trypsinization of sections from formalin fixed material increased the number of stainable cells substantially. No essential effect was seen on tissue fixed in Lillie's AAF and Bouin's fluid. In contrast trypsin treatment of sections from tissue fixed in Clarke's fluid and E--A completely destroyed the tissue. No differences were observed between different immunoglobulin classes examined as regards the effect of fixation time, fixatives and trypsinization.     

Rapid microwave fixation of human tissues for light microscopic immunoperoxidase identification of diagnostically useful antigens

Microwave (MW) energy permits rapid tissue fixation for light and electron microscopy but its effects on antigen preservation have not been fully evaluated. We, therefore, fixed three samples of human skin, uterus, and cervix, and two samples of human colon and breast by MW irradiation (5 to 8 seconds) during simultaneous immersion in a dilute aldehyde mixture (2% formaldehyde and 0.05% glutaraldehyde). For comparison, similar portions of each specimen were fixed in formalin. Specimens were processed routinely and embedded in paraffin for light microscopy. Sections from each specimen were stained with hematoxylin and eosin and, by immunoperoxidase techniques, for epithelial membrane antigen, leukocyte common antigen, S-100 keratin, carcinoembryonic antigen, and factor VIII-related antigen, the latter three with and without preliminary trypsinization. Colon sections were also stained for chromogranin. In all cases, light microscopic morphology was comparable for tissues fixed by the MW method and formalin-fixed specimens, as was immunostaining for epithelial membrane antigen, leukocyte common antigen, S-100 protein, and chromogranin. Formalin-fixed tissues required trypsinization for optimal detection of keratin, carcinoembryonic antigen, and factor VIII-related antigen. In contrast, trypsin-pretreatment was not necessary to demonstrate these antigens in MW-fixed specimens and, in fact, resulted in tissue digestion. We conclude that this MW fixation method provides a means for rapidly fixing tissues for immunoperoxidase staining while preserving excellent light microscopic morphology.