Detection of smoothelin expression in the urinary bladder is strongly dependent on pretreatment conditions: a critical analysis with possible consequences for cancer staging

Distinguishing urinary bladder muscularis propria (MP) from muscularis mucosae (MM) is crucial in bladder cancer staging. Immunohistochemical staining for the smooth muscle-specific protein smoothelin has been reported to be a robust marker for MP. The aim of this study was to investigate how smoothelin immunostaining in the bladder varies with pretreatment techniques and if it can be used to discriminate between MM and MP. Immunohistochemistry (IHC) for smoothelin was performed on nontumoral sections from 18 cystectomy specimens using three different pretreatment protocols. The immunoreactivity of MM, MP and blood vessels was scored semiquantitatively. Staining intensity depended strongly on the different pretreatment protocols used. Heat-induced epitope retrieval (HIER) in alkaline buffer resulted in the strongest staining with a moderate or strong immunostaining of the MP in 18/18 (100%) of cases, but in 11/18 (61%), the MM was moderately or strongly stained. HIER in acidic buffer resulted in a suboptimal staining of the MP. Enzymatic pretreatment resulted in absent or weak staining. In conclusion, smoothelin IHC is strongly dependent on epitope retrieval, and smoothelin staining did not discriminate reliably between MP and MM with any of the tested pretreatment protocols.     

High molecular weight cytokeratin antibody (clone 34betaE12): a sensitive marker for differentiation of high-grade invasive urothelial carcinoma from prostate cancer

AIMS: There is no well-established positive immunomarker for urothelial carcinoma. We evaluated the diagnostic utility of high molecular weight cytokeratin (HMWCK) antibody clone 34betaE12 in differentiating high-grade invasive urothelial carcinoma from prostate cancer. METHODS AND RESULTS: Formalin-fixed paraffin-embedded sections from 28 cases of high-grade invasive urothelial carcinoma (20 not otherwise specified (UC-NOS), eight with glandular differentiation) and 20 cases of poorly differentiated prostate carcinoma were immunostained with a monoclonal antibody to carcinoembryonic antigen (CEA), clone 85A12 and with HMWCK antibody clone 34betaE12 after microwave pretreatment or protease 24 predigestion. All cases of UC-NOS expressed HMWCK on 34betaE12 immunostaining after microwaving or enzyme predigestion. Immunoreactivity was intense and diffuse in all the cases after microwave pretreatment, whilst with enzyme predigestion immunoreactivity was sometimes patchy with <50% tumour cells positive in 20% of cases. In comparison with 34betaE12, 85A12 was insensitive with 15% of UC-NOS cases totally CEA-negative and <50% tumour cell immunoreactivity in 60% of cases. Rare positive cells were present in two (10%) cases of prostate cancer with monoclonal anti-CEA and 34betaE12 on microwaved sections, but all the cases were HMWCK-negative using 34betaE12 on sections pretreated by enzyme digestion. CONCLUSIONS: HMWCK antibody clone 34betaE12, particularly when used with microwave heat retrieval, is a very sensitive positive marker for high-grade invasive urothelial carcinoma.     

Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies.

AIMS--To evaluate the effect of microwave irradiation on the staining quality of a range of commonly used primary antibodies in archival, formalin fixed, paraffin wax embedded material, with emphasis on antibodies that have previously worked successfully only on frozen tissue. METHODS--Immunocytochemistry (streptavidin-biotin complex technique) was performed on histological sections of a range of normal and pathological tissues, after varying treatment with microwave irradiation. The staining quality of each antibody was compared with that achieved without prior treatment of the sections or after enzyme predigestion. RESULTS--Microwave irradiation permitted successful immunostaining with 20 antibodies that stained only frozen tissues before. The staining characteristics of 21 antibodies that were already known to stain formalin fixed, paraffin wax embedded material were improved. Another 39 antibodies did not show enhanced staining with microwave irradiation. The method preserves tissue morphology and produces more consistent staining than that achieved by enzyme predigestion with many antibodies. Microwave irradiation may also allow some primary antibodies to be used at higher working dilutions. The citrate buffer used in this study avoids the necessity of exposure to heavy metal salts. CONCLUSIONS--Microwave antigen retrieval represents an important technical advance within immunocytochemistry that will greatly increase the range of antibodies which can be used to study formalin fixed, paraffin wax embedded tissues.     

The effects of antibody clone and pretreatment method on the results of HER2 immunostaining in cytologic samples of metastatic breast cancer: A query and a review of the literature

The standardization and use of heat-induced epitope retrieval (HIER) is particularly important with immunohistochemical markers that direct the course of cancer treatment, such as Herceptin therapy. Increasingly, many laboratories are performing immunohistochemical analysis using various antibodies and methodologies for HER2/neu. We attempted to determine the effects of antibody clone and pretreatment methods on the interpretation of HER-2/neu staining in cytologic samples. Cell block sections from 54 cases of metastatic breast cancer (24 FNAs, 30 effusions) were analyzed for HER2 expression using antibodies to CB-11, TAB250, and A0485. Antibodies were analyzed with and without HIER. One pathologist using the FDA-approved scoring system for the HercepTest reviewed all slides in a blinded fashion. Five of fifty-four cases (9%) using CB-11 showed a significant increase in HER2 immunoreactivity using HIER (i.e. from 0/1+ to 2-3+). However, in twenty-nine of fifty-four cases (54%), the cytoplasmic background was significantly higher after HIER. With the A0485 antibody, two of fifty four cases (4%) showed a significant increase in immunoreactivity using HIER, while seventeen of fifty-four cases (31%) exhibited only more pronounced cytoplasmic staining. HIER pretreatment did not increase HER2 staining in any TAB250 stained sample, rather four of fifty-four cases (7%) showed a significant decrease in staining with HIER. We conclude that HIER may enhance membrane staining with the CB-11 and A0485 antibodies, but also increases cytoplasmic background. Loss of antigenicity is seen when HIER is used with TAB250.     

A molecular model of antigen retrieval using a peptide array

Even though antigen retrieval is highly denaturing, it paradoxically restores immunoreactivity after formalin fixation. It is unclear how this happens. We address this question using a peptide array to model formalin fixation and antigen retrieval. The peptides are linear stretches based on the native protein sequence, containing antibody epitopes of HER-2, estrogen receptor, progesterone receptor, and Ki-67. Of the 7 peptides, 6 retain their immunoreactivity after formalin fixation. However, if formalin fixation is performed in the presence of an irrelevant protein, immunoreactivity is abrogated, regardless of the peptides' amino acid composition. Fixation of an external protein around the antibody epitope prevents antibody binding. Antigen retrieval restores immunoreactivity. These findings demonstrate that native protein conformation is not relevant during antigen retrieval. Moreover, the loss and recovery of immunoreactivity associated with fixation and antigen retrieval, respectively, can be accounted for completely with a model of steric interference by adjacent proteins.     

Variations in immunohistochemical detection of p53 protein overexpression in cervical carcinomas with different antibodies and methods of detection

Immunocytochemical detection of p53 protein products in paraffin sections is possible with a number of antisera, monoclonal and polyclonal. Few corroborative results amongst different laboratories have been published due to variations in the antibodies, the fixation protocols, and the immunocytochemical methods applied. Antigen unmasking methods employing microwaves or proteolytic enzymes add to the disparity in the percentage of positive cases reported. In this study, paraffin sections of 55 cases of cervical carcinoma were immunostained with monoclonal antibodies p53-DO7 and p53-1801 (a) without section pretreatment, (b) with pronase digestion, and (c) with microwave antigen retrieval in citric acid buffer. Specimens fixed in buffered formalin required antigen unmasking to show positive staining. Pronase digestion caused false-negative immunostaining. Microwave pretreatment with p53-DO7 yielded 100 per cent positivity for p53 proteins but only 7/55 cases with more than 50 per cent positive cells. Monoclonal antibody p53-DO7 detected more positive cases than p53-1801. Immunostaining with antibodies to p53 proteins must be interpreted cautiously as variations in fixation, antibodies, and section pretreatment will significantly affect results.     

Immunostaining of cell preparations: A comparative evaluation of common fixatives and protocols

Immunostaining of cytologic preparations has been beset by problems of inconsistency, high background staining, and the requirement of different fixatives for different antigens. This study sought to identify a universal fixative and a simple fixation protocol suitable for a wide range of tissue antigens commonly employed for cytologic diagnosis. In an analysis of 23 fixation protocols involving acetone, acetone/methanol, acetone/formalin, glutaraldehyde, ethanol, methanol, and formal saline, fixation in 0.1% formal saline overnight at 27°C followed by 10 min fixation in 100% ethanol produced the most consistent and optimal preservation of immunoreactivity which could be further enhanced by pre-treatment with microwaves for epitope retrieval. Blocking of endogenous peroxidase was not necessary with this fixation protocol. Provided the smears were well air-dried (for at least 14 hr) prior to immersion in formal saline, there was no need to employ adhesive-coated glass slides. The smears could be kept at 27°C (room temperature) for at least 7 days and at −70°C for 5 wk without loss of immunoreactivity as air-dried smears or after fixation in formal saline. One hundred percent acetone and 100% ethanol produced good morphology and immunoreactivity but a high level of background staining, whereas acetone-based mixtures resulted in inconsistent immunostaining. Diagn Cytopathol 1996;15:167–174. © 1996 Wiley-Liss, Inc.     

Immunostaining of intermediate filament proteins in paraffin sections. Evaluation of optimal protease treatment to improve the immunoreactivity

Different protocols of protease (pepsin) treatment were compared in the immunostaining for intermediate filament (IF) proteins in formaldehyde-fixed and paraffin-embedded tissues. Without protease treatment, the immunoreactivity for all IF proteins was poor in such material. Appropriate pepsin pretreatment improved the immunoreactivity of formaldehyde-fixed tissues for all types of IF proteins, except for the 68 K neurofilament protein, which could not be immunostained with the antibody used. The optimal time for the pepsin treatment was varying in different tissues, and too long treatment caused progressive loss of immunostaining. Thus, for instance when demonstrating cytokeratins, renal adenocarcinomas were more sensitive to protease and needed a shorter treatment than other carcinomas. Therefore, a nonoptimal protease treatment protocol may cause false negative results and false cell type selective IF immunostaining. Prolonged fixation made it necessary to prolong the protease treatment. In tissues fixed up to four years in formalin, cytokeratin immunoreactivity could still be restored by a long pepsin treatment (up to 2-3 hours). For most tissues fixed for 24 hours in formaldehyde, an optimal protocol was the following: 0.05% pepsin (2 U/ml) in HCl, pH 1.8, at +37 degrees C for 20-30 minutes. The protease treatment did not produce false positive results. Alcohol-fixed material was good for IF immunostaining without any protease treatment, but such tissue blocks mostly lost the immunoreactivity during long term storage.     

Study of the microwave fixation using the immunohistochemical method]

We evaluated microwave fixation of formalin immersed and microwave irradiated kidney and liver tissues by keratin immunostain using the paraffin embedded section. In the microwave irradiated tissues, the formalin fixed areas and the alcohol fixed areas were clearly detected because; 1) Alcohol fixed tissues are easily digested by pepsinization but formalin fixed tissues are not, and 2) Formalin fixed tissues revealed intense keratin staining after pepsinization, whereas intense keratin staining were noted in alcohol fixed tissues without preliminary pepsinization. The microwave irradiation fixed completely the tissues in about 20 sec. A thin, formalin-fixed layer was observed in the periphery of the tissues. A thin, alcohol-fixed layer was observed beneath the peripheral formalin fixed layer in some specimens. This area was thought to have been fixed during the dehydration of the paraffin blocks. However with microwave fixation, most of the central areas differed from those of the formalin or alcohol fixed areas. They showed sharp contrast on hematoxylin-eosin staining, conspicuous cellular membrane and only slight cellular shrinkage and swelling. Unlike the formalin and alcohol fixed tissues, the intensity of keratin staining was independent of preliminary pepsinization and keratin staining was observed. Therefore, the central areas were thought to be fixed by the microwave irradiation. This fixation method seems to be useful for immunostaining.     

High quality of DNA retrieved for Southern blot hybridization from microwave-fixed, paraffin-embedded liver tissues

To overcome the degradation problem encountered in DNA extracted from formalin-fixed, paraffin-embedded tissue blocks, several methods of tissue fixation were examined in order to improve the quality of the DNA recovered for use in nucleic acid analysis. The fixation methods included formalin fixation alone, alcohol fixation alone, and microwave fixation with tissues immersed in phosphate-buffered saline (PBS), alcohol, or formalin. Unfixed fresh frozen tissue served as the control. Using hepatitis B virus (HBV) DNA sequences and the type I human procollagen gene as markers and liver tissue as a target, microwave fixation, with formalin omitted, not only preserved the DNA very well, but also the labile viral antigen. Both high molecular weight-integrated and free-form HBV DNAs were well preserved, and suitable for polymerase chain reaction and Southern blot analysis. The restriction enzyme fragment pattern of DNA recovered from these paraffin blocks was identical to that of unfixed fresh frozen tissue. Microwave fixation also preserved the labile preS2 epitope of the hepatitis B surface antigen (HBsAg) considerably better than formalin. These results suggest that microwave fixation is superior to routine formalin fixation for the preservation of excellent quality of genomic and viral DNAs for nucleic acid hybridization analysis. Alcohol, often used for nucleic acid purification, was also a good fixative for preserving DNA and the antigenicity of the labile antigen, especially when carried out in combination with microwave fixation.     

Tissue preparation for immunocytochemistry.

AIMS: To investigate the effect of tissue preparation on immunostaining and to establish whether there is a standard tissue preparation schedule that allows optimal demonstration of all antigens. METHODS: Blocks of tonsil were subjected to variations to a standard fixation, processing, and section preparation schedule. The sections were stained with five antibodies-L26 (CD20), UCHL1 (CD45RO), CD3, vimentin, and anti-kappa light chain--using the streptavidinbiotin immunostaining technique. When further investigation was necessary, other tissues and antibodies were used and where weak immunostaining was obtained the use of microwave pretreatment to improve staining was tested. RESULTS: Several factors involved in fixation were found to affect immunoreactivity. These included the duration, pH, and type of fixative used. In tissue processing only temperature and the duration of the dehydration and wax infiltration steps affected immunoreactivity. Of all the factors investigated, the temperature and duration of the section drying had the greatest effect. In contrast, long term storage of cut sections before immunostaining had no effect on the reactivity of the antibodies tested. Antibodies were found to be affected by alterations to tissue preparation by varying degrees, UCHL1 and vimentin being the most susceptible to changes in fixation and L26 to changes in processing. Where weak staining occurred, microwave pretreatment was generally found to eliminate the problem. CONCLUSIONS: There is no standard tissue preparation schedule for the optimal demonstration of all antigens. Factors involved in all aspects of tissue preparation can affect immunoreactivity, so it is important that precise details of the preparation schedule are given when reporting immunocytochemical studies, rather than using the general term "routinely fixed and processed".     

Sporicidal activity of chemical and physical tissue fixation methods.

AIMS: The effects of alcohol based fixation and microwave stimulated alcohol fixation were investigated on spores of Bacillus stearothermophilus and Bacillus subtilis (var. niger). METHODS: Spores were exposed to 10% formalin, or different concentrations of various alcohol containing fixatives (Kryofix/Spuitfix). Adequate controls were also set up in conjunction with the test solutions. The spores were immersed with and without adjunctive microwave stimulation in the various solutions tested. Possible surviving spores were recovered in revival broth and after incubation, and Gram staining viable counts were performed. RESULTS: Alcohol based fixatives did not have a sporicidal effect on B stearothermophilus or B subtilis (var. niger) spores, and microwave stimulated alcohol fixation at 450 W and up to 75 degrees C did not have a sporicidal effect. CONCLUSIONS: When alcohol based fixatives are used for fixation, precautions should be taken with the material thus treated, as it may contain viable spores or other pathogens, which are destroyed after 24 hours of formalin treatment. Of the physicochemical methods tested involving microwaving, none was successful in eliminating viable spores from the test material.     

Quantitative evaluation of HER-2/neu status in breast cancer by fluorescence in situ hybridization and by immunohistochemistry with image analysis

We correlated quantitative results obtained in 40 invasive breast cancer cases for HER-2 gene amplification by fluorescence in situ hybridization with protein expression by immunohistochemical studies with computer-assisted image analysis. Fluorescence in situ hybridization (FISH) results were quantified as the mean number of fluorescent signals per nucleus, and immunohistochemical slides were read by semiquantitatively assessing membranous immunostaining intensity in tumor cells vs nonneoplastic breast tissue or quantitatively evaluated by image analysis. We found high correlation between immunohistochemical results by semiquantitative scoring and by image analysis. FISH results correlated with immunohistochemical results moderately when the staining intensity of only tumor cells was assessed and significantly better when the difference in staining intensity between tumor cells and nonneoplastic breast tissue was assessed. The correlation with FISH results was further improved when immunohistochemical study was combined with heat-induced epitope retrieval (HIER). Although FISH and immunohistochemical studies assess different aspects of the HER-2/neu gene (amplification vs overexpression), we found good correlation between the diagnostic techniques. The correlation was best when immunohistochemical studies were combined with HIER and assessed as the difference between tumor cells and nonneoplastic breast tissue.     

加热抗原修复对内源性抗生物素蛋白结合物的影响及其对策

目的：深入研究加热抗原修复对内源性抗生物素蛋白结合物（EABA）的影响程度和范围,以及探讨消除EABA对免疫组织化学染色干扰的对策或方法。方法：采用高效、快捷的组织芯片技术、微波加热处理法、免疫组织化学SP染色法及蛋清液封闭法,对甲醛固定石蜡包埋的76种（102份）人体正常组织和88种（577份）人体肿瘤组织以及4种（80份）大鼠正常组织标本进行系统性EABA检查,同时对9种（15份）人体冷冻组织进行EABA检查。结果：（1）冷冻组织中存在EABA;（2）经甲醛固定石蜡包埋后组织中的EABA被封闭;（3）加热抗原修复可造成EABA暴露;（4）EABA暴露的强度各不相同,强者可到强阳性;（5）EABA在组织中的分布形式,即有散在分布也有弥漫分布,主要以颗粒状形式存在于胞质中;（6）EABA广泛存在于上皮源性组织,特别是腺上皮组织（包括正常组织和肿瘤组织）,亦存在于部分非上皮组织;（7）EABA暴露的强弱与修复液亦有关,EGTA（pH9.0)的暴露能力较柠檬酸（pH6.0)和乙二胺四乙胺四乙酸二钠（pH8.0)更强;（8）加热抗原修复暴露的EABA可以被20％蛋清液封闭。结论：加热修复可使甲醛固定石蜡包埋组织中的EABA重新暴露,暴露的EABA可广泛见于人体正常和肿瘤组织细胞中,也见于大鼠正常肝、肾、肺、胰组织中,因而可能对正常免疫组织化学染色造成干扰。暴露的EABA可被蛋清液封闭,或采用非生物素检测系统,达到消除EABA对正常免疫组织化学染色干扰的目的。     

The effects of glyoxal fixation on the histological evaluation of breast specimens

Glyoxal (GL), a non-formalin-containing aldehyde tissue fixative, is advocated as a superior fixative that is environmentally safe and lacks the purported carcinogenic health hazards associated with formalin use. In addition, it is advertised as requiring no antigen retrieval before immunohistochemical staining. We compared GL fixation to standard formalin fixation of breast specimens removed for microcalcifications or breast tumors. Although the hematoxylin and eosin morphology of GL-fixed and formalin-fixed tissues was equivalent, detection of microcalcifications in GL-fixed breast specimens was hampered by loss of basophilia, likely due to increased calcium solubility in glyoxal. Moreover, estrogen receptor detection in GL-fixed specimens was diminished compared to formalin and did require antigen retrieval.     

A molecular mechanism of formalin fixation and antigen retrieval

Despite the popularity of antigen-retrieval techniques, the precise molecular mechanism underlying the process remains enigmatic. We examined the molecular features underlying the loss of immunoreactivity following formalin fixation, with subsequent recovery by antigen retrieval. To do this, we first created a molecular model using short peptides that mimic the antibody-binding site of common clinical protein targets. The advantage of this model is that we know the amino acid sequence in and around the antibody-binding site. We observed that some, not all, of the peptides exhibited the formalin-fixation and antigen-retrieval phenomenon. Other peptides did not lose their ability to be recognized by antibody, even after prolonged incubation in formalin. A third, intermediate group exhibited the formalin-fixation and antigen-retrieval phenomenon only if another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicates that fixation and antigen retrieval are associated with a tyrosine in or near the antibody-binding site and with an arginine elsewhere, implicating the Mannich reaction as important in fixation and antigen retrieval.     

Immunoglobulin light chain staining in paraffin-embedded tissue using a heat mediated epitope retrieval method

We have compared light chain immunohistochemistry in reactive lymphoid tissue and a series of paraffin-embedded B-cell lymphomas using standard trypsin digestion with a heat mediated epitope retrieval method. Fifty-seven B-cell lymphomas (18 high grade, 29 low grade and 10 cases of nodular lymphocyte predominance Hodgkin's disease), two reactive lymph nodes and eight tonsils fixed for known times between 12 h and 2 years were studied. Paraffin-embedded tissue was stained with polyclonal anti-kappa and anti-lambda antibodies. For each antibody staining was performed on two sections, one treated with trypsin digestion and one with microwave heating. Sections were scored from 0 to +++ with 0 representing poor staining and +++ excellent staining. A score of ++ was considered satisfactory. Light chain restriction was recorded if present. Satisfactory staining was obtained in 34/59 cases using trypsin digestion and 56/59 cases using heat retrieval. Light chain restriction was demonstrated in 32/57 (56%) B-cell lymphomas using trypsin digestion and 52/57 (91%) using heat retrieval. Satisfactory staining was obtained in tonsils fixed for up to 48 h using trypsin digestion and up to 2 years using heat retrieval. We have shown that for light chain immunostaining a heat mediated epitope retrieval method produces more consistent and satisfactory results and is effective over a greater range of fixation times than traditional trypsin digestion.     

CD31 (JC70) expression in plasma cells: an immunohistochemical analysis of reactive and neoplastic plasma cells.

AIMS: To investigate the immunohistochemical expression of CD31 (JC70) in normal and neoplastic plasma cells. METHODS: Plasma cells in bone marrow biopsies and extramedullary locations were examined. All extramedullary biopsies were formalin fixed and paraffin embedded. The bone marrow biopsies were fixed in formal acetic acid and embedded in paraffin wax. Twenty multiple myelomas (12 bone marrow and eight extramedullary deposits), 10 extramedullary plasmacytomas, and 30 biopsies with reactive plasma cells (10 bone marrow, 20 extramedullary biopsies) were stained with anti-CD31 (JC70) using the streptavidin-biotin detection system with diaminobenzidine as a chromogen. Antigen retrieval in bone marrow biopsies was achieved by pressure cooking. In all other biopsies, antigen retrieval was achieved by microwave pretreatment. RESULTS: All 20 extramedullary cases with reactive plasma cells showed intense membrane staining. Focal staining was detected in reactive plasma cells in bone marrow biopsies. Five of 10 plasmacytomas showed membrane staining. None of the cases of multiple myeloma, either medullary or extramedullary, showed any immunoreactivity for CD31. CONCLUSIONS: CD31, a member of the immunoglobulin supergene family of cell adhesion molecules, is strongly expressed in extramedullary reactive plasma cells, focally in bone marrow reactive plasma cells, and occasionally in extramedullary plasmacytomas.     

Immunohistochemistry of tissue prepared by a molecular-friendly fixation and processing system.

A recently introduced histologic fixative (Universal Molecular Fixative [UMFIX]) has been shown to preserve macromolecules in tissue at ambient temperature. When UMFIX-exposed tissues are processed by a formalin-free, microwave-assisted rapid processing system, the resulting paraffin blocks retain good histomorphology and intact nucleic acids suitable for expression microarray analysis. Because UMFIX may be used as an alternative to formalin, the authors set out to study the effect of this new fixation and processing system on immunohistochemistry (IHC) by analyzing a range of human neoplastic and non-neoplastic specimens. Parallel slices from surgically removed specimens were fixed in formalin and UMFIX and processed in a rapid microwave-assisted tissue processor. IHC was performed following routine procedures. The staining for those antibodies that normally required antigen retrieval was carried out with and without that step. The intensity and pattern of reactions were compared in 144 tissue samples fixed by the two methods using 70 monoclonal and polyclonal antibodies. The intensity of IHC reactions for most cytoplasmic antigens was generally equal or stronger in UMFIX tissues. This was particularly true with intermediate filaments and HercepTest, where the antigen retrieval step became unnecessary. Conversely, there was a decrease in the intensity of reactions for HepPar1, bcl-2, and three nuclear antigens (Ki-67, TTF-1, and estrogen receptor). Increasing their exposure times optimized the sensitivity of the latter four antibodies. The study shows that IHC staining results of tissues fixed in UMFIX and processed by the microwave-assisted system are comparable to those obtained on formalin-fixed, similarly processed specimens. There is an enhancement of the sensitivity of few antibodies in UMFIX-exposed tissue, rendering antigen retrieval unnecessary. This increased sensitivity may be due to the effect of eliminating formalin from fixation and processing or the microwave energy.