Antigen Retrieval Method Research Articles

Inflammatory response to chronic otitis media in DiGeorge syndrome: a case study using immunohistochemistry on archival temporal bone sections.

Immunohistochemical analyses were conducted on archival celloidin-embedded human temporal bone sections from an 8-month-old boy with chronic otitis media and DiGeorge syndrome. We employed antigen retrieval methods with saturated sodium hydroxide-methanol solution, microwave incubation, and proteolytic treatment to demonstrate the distribution of T-lymphocytes, B-lymphocytes, macrophages, and intercellular adhesion molecule 1 (ICAM-1) expression in the middle ear. B-lymphocytes and macrophages were observed predominantly within the middle ear mucosa. T-lymphocytes were rare. Further, ICAM-1 was expressed in the vascular endothelium of the lamina propria, as well as infiltrating mononuclear cells. This suggests that the expression of ICAM-1 can be induced in the middle ear with otitis media, even if T-lymphocytes are depressed in a cell-mediated immunodeficiency disorder such as DiGeorge syndrome.

The visualization of a new class of traumatically injured axons through the use of a modified method of microwave antigen retrieval.

Antibodies to the amyloid precursor proteins (APP) have become routine markers for detecting traumatically induced axonal injury (AI) in animals and man. Unfortunately, the techniques used to visualize these proteins are not compatible with routine electron microscopic (EM) analysis. In the current communication, we describe a method for the ultrastructural visualization of antibodies to APP and, using this method, we identify a previously unrecognized population of traumatically injured axons. Rats were subjected to an impact acceleration traumatic brain injury and allowed to survive 30 min to 3h postinjury. The animals were then perfused, their brains sectioned on a vibratome and the sections prepared for immunocytochemistry using a computer-controlled microwave capable of temperature regulation. The use of temperature-controlled microwave energy unmasked APP antigenic epitopes without sacrificing ultrastructural detail. The APP antibody was found in two distinct populations of reactive axons that differed in size, morphology, location, and temporal progression. Comparable to previous descriptions, one population showed traumatically related reactive changes that led to swelling and disconnection. The other population, however, revealed unanticipated changes reflected in nodal and paranodal swelling of small continuous fibers that showed no evidence of disconnection during the time periods assessed. These studies provide new insight into the complexity of the pathobiology of AI, while describing a novel approach for enhancing APP immunoreactivity at the EM level.

Image Analysis for Quantitative Evaluation of Antigen Retrieval Efficacy Demonstrates Increased Detection of P-glycoprotein in Overfixed Cells but Decreased Detection in Optimally Fixed Cells

We developed a protocol to objectively quantify, via image analysis, the results of antigen retrieval (AR) maneuvers on immunohistochemical results for detection of P-glycoprotein (P-gp) in paraffin embedded test samples. Two carcinoma cell lines, KB 3-1 and KB V-1 (previously described non-expresser and high expresser of P-glycoprotein, respectively), were harvested, fixed for 4, 8, 12, 24 and 48 hr in formalin, placed in collodion bags, embedded into a single paraffin block, subjected to various antigen retrieval techniques, and stained with the anti-P-gp antibody C494. The amount of immunostaining was quantitated with the CAS 2.0 cell measurement program and optical density ratios were compared between the positive and negative cell lines. In overfixed cells (48 hr, 10% formalin), AR with microwaves and/or trypsin or other enzymes proved effective in optimizing P-gp detection as evidenced by an increased optical density ratio. In contrast, quantitation of staining of optimally fixed cells (4 hr in 10% formalin) demonstrated that AR decreased the optical density ratio. A postfixation step after the primary antibody also served to decrease the optical density ratio, via an increase in noise in the non-expressing cell. The use of optical density analysis by image computerization allowed objective, exquisitely sensitive, quantitative assessment of AR methods. (The J Histotechnol 22:9, 1999)

Image Analysis for Quantitative Evaluation of Antigen Retrieval Efficacy Demonstrates Increased Detection of P-glycoprotein in Overfixed Cells but Decreased Detection in Optimally Fixed Cells

We developed a protocol to objectively quantify, via image analysis, the results of antigen retrieval (AR) maneuvers on immunohistochemical results for detection of P-glycoprotein (P-gp) in paraffin embedded test samples. Two carcinoma cell lines, KB 3-1 and KB V-1 (previously described non-expresser and high expresser of P-glycoprotein, respectively), were harvested, fixed for 4, 8, 12, 24 and 48 hr in formalin, placed in collodion bags, embedded into a single paraffin block, subjected to various antigen retrieval techniques, and stained with the anti-P-gp antibody C494. The amount of immunostaining was quantitated with the CAS 2.0 cell measurement program and optical density ratios were compared between the positive and negative cell lines. In overfixed cells (48 hr, 10% formalin), AR with microwaves and/or trypsin or other enzymes proved effective in optimizing P-gp detection as evidenced by an increased optical density ratio. In contrast, quantitation of staining of optimally fixed cells (4 hr in 10% formalin) demonstrated that AR decreased the optical density ratio. A postfixation step after the primary antibody also served to decrease the optical density ratio, via an increase in noise in the non-expressing cell. The use of optical density analysis by image computerization allowed objective, exquisitely sensitive, quantitative assessment of AR methods. (The J Histotechnol 22:9, 1999)

Immunoreactivity of a new CD5 antibody with normal epithelium and malignant tumors including thymic carcinoma.

CD5, first recognized on subsets of lymphocytes, also is detected in thymic carcinoma but not in thymoma or other malignant tumors. We studied CD5 expression in 73 cases of malignant tumors of various organs, 22 cases of thymoma, and 7 cases of thymic carcinoma by immunohistochemistry using the new monoclonal anti-CD5 antibody, NCL-CD5-4C7, with a pressure cooker antigen retrieval method. All cases of thymic carcinoma showed positive staining for CD5, predominantly on the cell membrane. Two of 4 cases of atypical thymoma also showed focal positivity, whereas the other types of thymoma were negative. CD5 was detected in cases of malignant tumors other than squamous cell carcinoma and in the normal epithelium of their counterparts. Squamous cell carcinomas of various organs were negative for CD5. Malignant mesothelioma showed peculiar intracytoplasmic staining in contrast to the other tumors. The NCL-CD5-4C7 positivity in thymic epithelial tumors may support the hypothesis suggesting progression of atypical thymoma to thymic carcinoma. NCL-CD5-4C7 may be useful in the differential diagnosis of mediastinal tumors, especially between thymic carcinoma and metastatic squamous cell carcinoma of various primary sites, and for distinguishing malignant mesothelioma from adenocarcinoma of the lung by the different staining pattern.

Expression of c-erbB-2 oncoprotein in human thyroid tumours.

c-erbB-2 expression has been found to be a potential marker of aggressive biological behaviour in some tumours, but the role played by this oncoprotein in the development and maintenance of thyroid tumours is still controversial. Therefore our objective was to determine whether c-erbB-2 was overexpressed in a large retrospective series of human thyroid tumours, including both from follicular and C-cell differentiation. We have studied 67 thyroid tumours (10 follicular adenomas, 11 follicular carcinomas, three anaplastic carcinomas, 25 papillary carcinomas and 18 medullary carcinomas and 16 metastases) by immunohistochemistry using an antigen retrieval method for paraffin-embedded material and a specific polyclonal antibody against the intracytoplasmic part of c-erbB-2 oncoprotein. There are marked differences in the pattern of c-erbB-2 immunoreactivity depending on the type of thyroid tumour. Thus, no expression of the oncoprotein has been found in follicular adenomas, follicular carcinomas and anaplastic carcinomas, but 52% of papillary carcinomas (membranous and diffuse cytoplasmic patterns) and all medullary carcinomas (granular cytoplasmic pattern) are immunopositive. Our results indicate that overexpression of c-erbB-2 oncoprotein is easily identifiable by immunohistochemistry in paraffin sections of certain thyroid tumours after applying an antigen retrieval method. This study suggests that c-erbB-2 oncoprotein may play some role in disease progression in papillary and medullary thyroid carcinomas, but the significance of the different immunohistochemical patterns merits further investigations.

C-erbB-2 protein expression in oesophageal squamous epithelium from oesophageal squamous cell carcinomas, with special reference to histological grade of carcinoma and pre-invasive lesions

C-erbB-2, an oncogene, is member of the growth factor receptor family. Its role in activation of oesophageal squamous cell carcinoma is poorly understood. The aim of this study was to evaluate the part played by c-erbB-2 in oesophageal squamous cell carcinoma in Hong Kong Chinese patients. We examined the expression of the c-erbB-2 oncoprotein in 104 oesophageal squamous cell carcinomas from 89 men and 15 women, ranging in age from 41 to 89 years (mean 63). C-ercB-2 expression was studied with monoclonal antibody, using an antigen retrieval method. Focal c-erbB-2 membrane staining was present in 10 (10%) of 104 squamous cell carcinomas. Staining was also noted in the adjacent dysplastic epithelium (n=2) and non-tumour inflamed epithelium (n=2). In carcinomas, the c-erbB-2 membrane staining was identified only in superficial well-differentiated tumour cells and the expression did not predict biological behaviour. We conclude that the c-erbB-2 oncoprotein is expressed in a portion of oesophageal squamous cell carcinomas and precursor lesions. This suggests that c-erbB-2 activation plays a certain role, mostly probably during the early stages, in carcinogenesis in oesophageal squamous cell carcinomas from Hong Kong Chinese patients.

Nucleolar localization of BRCA1 protein in human breast cancer.

Both a rabbit polyclonal BRCA1 antibody, K-18, and a mouse monoclonal BRCA1 antibody, AP 16, produced nucleolar epithelial cell staining on frozen tissue sections of human infiltrating mammary carcinomas. There was much less BRCA1 antibody staining in normal tissues; however, 2 intraductal tumors and a papilloma, found in proximity to the carcinomas showed considerable nucleolar immunoreactivity. MCF-7 cells fixed in methanol and immunostained with the same two antibodies also revealed nucleolar staining, however, after 4% paraformaldehyde fixation for three minutes, there were many fewer nuclei stained. Antigen retrieval methods on formalin-fixed, paraffin-embedded specimens produced tumor cell cytoplasmic staining with AP 16 and nuclear staining in both tumor and normal epithelial cells with another BRCA1 monoclonal antibody, SG 11.

Antigen Retrieval in Formaldehyde-Fixed Human Brain Tissue

Microwave-stimulated antigen retrieval has become a widely accepted method in both pathology and research laboratories. Since the introduction of the method in 1991, many groups have tried to optimize and standardize it. This review describes the present state of the art. A standard method for microwave-stimulated antigen retrieval in formaldehyde-fixed paraffin-embedded and nonembedded tissue is presented that results, in general, in very good staining for antibodies used in neuroscience. However, there are still a few antigens that are retrieved not at all or not in an optimal manner. Factors of importance for microwave antigen retrieval are the pH of the retrieval solution and, related to the pH, the temperature and duration of heating. These factors are discussed.

Improved detection of CD5 epitope in formalin-fixed paraffin-embedded sections of benign and neoplastic lymphoid tissues by using biotinylated tyramine enhancement after antigen retrieval.

To evaluate the effectiveness of the immunohistochemical staining of B- and T-cell lymphomas with Leu-1 (clone L17F12 CD5 antibody, Becton Dickinson, San Jose, Calif) in formalin-fixed paraffin-embedded sections, we stained 12 specimens reflecting cases of chronic lymphocytic leukemia/small lymphocytic lymphoma, 7 of mantle cell lymphoma, 13 of T-cell lymphomas, and 9 of various B-cell neoplasms that do not ordinarily express CD5, using a streptavidin-horseradish peroxidase method with biotinylated tyramine enhancement after antigen retrieval. We were able to detect CD5 reactivity of neoplastic cells in 9 (75%) of 12 cases of chronic lymphocytic leukemia, 6 (86%) of 7 cases of mantle cell lymphoma, and 13 (100%) of 13 of the T-cell lymphomas. B-cell neoplasms (9/9) not typically associated with CD5 expression showed no reactivity of tumor cells. We conclude that the Leu-1 (CD5) antibody, routinely used for cryopreserved tissues, is also effective in formalin-fixed paraffin-embedded sections using an antigen retrieval and streptavidin-horseradish peroxidase method with biotinylated tyramine.

P53 protein immunohistochemical expression in colonic adenomas with and without associated carcinoma

Objectives: P53 protein immunohistochemical (IHC) expression was investigated in a series of colonic adenomas and carcinomas to determine the p53 immunohistochemical expression of adenomas in general compared with carcinomas, the difference in staining pattern between adenomas with associated carcinoma and those without associated carcinoma, and the difference in p53 staining in the usual adenomas (low-grade dysplasia) compared with those harboring high-grade dysplasia. Methods: The study involved a series of 20 adenomas without concurrent carcinoma (group 1 adenoma), 29 adenomas with concurrent carcinoma (group 2 adenoma), and 20 carcinomas. Sections of the paraffin-embedded tissues were stained with DO-7 p53 monoclonal antibody after microwave antigen-retrieval method. Cases with nuclear staining in ≥ 20% of the tumor cells were considered positive. Results: Analysis of results showed that 65% of carcinomas and 37% of all adenomas were reactive with p53 IHC staining ( p = 0.03). With respect to the adenomas, 30% of group 1 and 41% of group 2 adenomas were reactive for p53 protein ( p = 0.42). Conclusions: Our data demonstrate a statistically significant higher p53 expression rate in colonic carcinomas than in adenomas, and that adenomas with concurrent carcinomas are more frequently p53 positive than those without concurrent carcinoma, but this was not statistically significant. Also, p53 expression is more frequent and intense in adenomas with high-grade dysplasia (10/20, 50%) than in ordinary adenomas with low-grade dysplasia (8/29, 28%), which suggests a strong correlation between the degree of dysplasia in colonic neoplasia and p53 expression pattern.

Multiple regression analysis for assessing the growth of small hepatocellular carcinoma: the MIB-1 labeling index is the most effective parameter.

The aim of this study was to clarify whether histological parameters reflected tumor aggressiveness in patients with hepatocellular carcinoma (HCC). The tumor volume doubling times (TVDTs) of 21 HCCs, less than 3 cm in diameter at the start of the observation period, were calculated in 21 patients in whom the natural progression of the lesion was observed by ultrasonography. Paraffin-embedded sections were prepared from samples obtained by ultrasound-guided fine-needle liver biopsy at the end of the observation period. The histological parameters examined were the MIB-1 labeling index (LI), for which we performed immunohistochemical staining with the MIB-1 monoclonal antibody, using an antigen retrieval method; the nucleo-cytoplasmic (N/C ratio), cellularity, and the nuclear form factor (NFF), were calculated with an imaging analyzer. We performed multiple regression analysis for estimating the growth of small HCCs. With the N/C ratio (0.154 +/- 0.068; mean +/- SD), cellularity (453 +/- 21.8 cells/10(4) microm2), NFF (1.150 +/- 0.096), and degree of HCC differentiation as independent variables, only the MIB-1 LI (11.8 +/- 6.1%) showed a significant correlation with TVDT (207.5 +/- 162.6 days) (r = -0.658; P < 0.05). Compared to the conventional indices of histological atypism tested, i.e., N/C ratio, cellularity NFF, and degree of HCC differentiation, only MIB-1 LI was significantly correlated with small HCC growth rate. The MIB-1 LI may therefore be a simple and useful index of tumor aggressiveness.

Immunohistochemistry of p53 in sinonasal inverted papilloma and associated squamous cell carcinoma.

Gene mutation of p53 is documented in head and neck cancer. No reports exist relating human sinonasal inverted papilloma (IP) and its transformation to squamous cell carcinoma (SCC). Using immunohistochemical techniques improved by an antigen retrieval method, p53 protein was analyzed in the following subjects: 16 IP, 12 SCC arising in IP, and 15 SCC alone subjects. Both the intensity (PI) and rate (PR) of positive p53 immunostaining were evaluated using a quantitative AutoCAD program. We correlated p53 protein expression with clinical features. p53 immunoreactivity is significantly lower in IP than in SCC specimens. Both the IP and SCC portions of IP synchronous SCC subjects showed similar p53 protein expression. SCC arising in IP shows a lower p53 immunoreactivity than SCC alone. Alteration of p53 protein may have an important role in the early stages of IP malignant transformation and prove a potential biomarker for risk assessment. Low p53 immunoreactivity, indicating the presence of a wild-type p53, may correlate with the prognosis for SCC arising in IP. However, further clinical trials are required to investigate this possibly worthwhile prognostic marker.

Assessment of p53 Protein Expression in Normal Mucosa and Benign and Malignant Lesions of the Nasal Cavity

p53 gene mutation is documented in head and neck cancer. No reports exist relating this mutation to normal mucosa or benign and malignant lesions of the nasal cavity. We investigate p53 overexpression using immunohistochemical techniques improved by an antigen retrieval method. p53 protein was analyzed in the following cases: normal, benign [papilloma and inverted papilloma (IP)] and malignant [squamous-cell carcinoma (SCC) arising in IP, SCC alone, adenocarcinoma and small-cell carcinoma]. Both the intensity and rate of positive p53 immunostaining were evaluated using a quantitative AutoCAD program. Overexpression of p53 protein was not identified in normal mucosa, benign or premalignant lesions; however, approximately 60% is correlated to nasal cancer. p53 overexpression correlates with heavy smoking. Both the IP and SCC portions of SCC synchronous with IP showed similar p53 immunoreactivity. SCC arising in IP shows a lower p53 immunoreactivity than SCC alone. Thus, smoking along with a p53 mutation may be a mutagenic agent in nasal cancers. Alteration of the p53 protein may play an important role in the early stages of the malignant transformation of IP. A low p53 immunoreactivity indicates the presence of wild-type p53 protein. This may show a better response to radiation therapy yielding a better prognosis for SCC arising in IP compared to SCC alone. However, further clinical trials are required to investigate this possibly worthwhile prognostic marker.

Immunohistochemical Detection of Progesterone Receptors in Archival Breast Cancer

The progesterone receptor (PR) is an important marker of response to endocrine agents in breast cancer. Immunohistochemical demonstration of PR in formalin fixed tissue has previously proved difficult, and heat pretreatment is considered necessary to retrieve the antigen. There are few data on the effectiveness of autoclaving in unmasking PR, however, and it is not known whether all PR epitopes are equally unmasked. The objectives of this study were to compare the efficacy of autoclaving and microwaving to retrieve PR antigen in archival breast tumors, to determine whether there is an epitope-dependent variability in the pretreatment required, and to examine different slide types and adhesives to reduce the problem of section loss frequently associated with these procedures. Paraffin embedded sections were cut at 2 or 4 μm, mounted onto various slide types with or without the addition of adhesive, and heat pretreated prior to immunoperoxidase staining. Whereas PR immunoreactivity was clearly demonstrated in tissue after both autoclaving and microwaving, autoclaving produced a significantly stronger staining intensity under the conditions used in this study. The duration of autoclaving required to reveal PR fully differed for different epitopes examined. In the absence of heat pretreatment, PR was not detected. Section retention was improved by the use of adhesives and by cutting tissue at 2 μm. Maximum retention was obtained using positively charged slides coated with Mayer albumen adhesive. We conclude that for maximal tissue preservation autoclave pretreatment is the preferred method of PR antigen retrieval from archival breast tumors, that there is epitope-dependent variability in pretreatment required, and that section loss during this procedure can be minimized by choice of slide type, the use of adhesive, and by cutting sections at 2 μm.

Retinoblastoma Susceptibility Gene Product Retrieval in Formalin-fixed, Paraffin-embedded Tissue: A Heating Method for Enhancing Immunohistochemical Staining.

We examined immunohistochemical methods to visualize the retinoblastoma susceptibility gene product (RB) in formalin-fixed, paraffin-embedded sections of the U2-OS and Saos-2 human osteosarcoma cell lines. Immunocytochemical studies showed clear staining with an anti-RB antibody in 88% of the smeared U2-OS cells and in none of the smeared Saos-2 cells. Antigen retrieval methods evaluated included microwaveoven and autoclave pretreatment of paraffin-embedded sections immersed in Dulbecco's phosphate buffered saline (pH 7.4) prior to incubation with the primary antibody. Strong and discrete staining of U2-OS cell nuclei for RB was seen after autoclave treatment, stronger than that seen following microwave-oven heating. Moreover, neither microwave-oven nor autoclave pretreatment produced positive staining in the nuclei of Saos-2 cells. Therefore, tissues previously considered unsuitable for immunohistochemical analysis of the RB can be studied after autoclave treatment.

Significant loss of pyramidal neurons in the angular gyrus of patients with Huntington's disease

The primary site of pathology in Huntington's disease (HD) is the caudate nucleus. However, cortical changes are also commonly reported. While many researchers have studied pathology in the frontal lobe, little attention has been paid to posterior cortical regions. The aim of this study is to examine pathology in the parietal lobe in patients with HD as it has specific projections to the caudate nucleus. Post-mortem brain tissue was obtained from HD patients with both a positive family history and clinicopathological diagnosis (n = 6; Vonsattel grades 2-4) as well as from neurologically normal controls (n = 6). The angular gyrus of the parietal lobe was sampled and cellular quantification of SMI-32 immunohistochemically detected pyramidal neurons performed. Cortical blocks were sectioned at 50 microns on a cryostat and stained immunohistochemically using antigen retrieval methods and peroxidase visualization. HD subjects had noticeable histological changes including smaller neurons and a disruption of cortical laminar pattern. Quantification using a point counting method to find the areal fraction of immunoreactive neurons revealed a severe loss of pyramidal neurons in the angular gyrus of HD subjects compared with controls (reduced on average to 55% of mean control values, P = 0.038 using the Mann-Whitney U-test). This striking cortical pathology suggests that HD may preferentially target posterior cortical regions, particularly the angular gyrus which has a significant projection to the caudate nucleus in primates.

Differential expression of N-cadherin in pleural mesotheliomas and E-cadherin in lung adenocarcinomas in formalin-fixed, paraffin-embedded tissues

The differential diagnosis of pleural mesotheliomas and lung adenocarcinomas presents a continued challenge in the practice of surgical pathology. Paraffin immunohistochemistry (IHC) using different panels of antibodies can be helpful in some cases, but, as yet, no antigen is expressed specifically in mesotheliomas nor in adenocarcinomas. Using well characterized monoclonal antibodies (MAb) that recognized distinct mesenchymal and epithelial adhesion proteins, N-cadherin (13A9 MAb) and E-cadherin (E9 MAb), respectively, we found previously that in frozen-section IHC mesotheliomas and adenocarcinomas had distinct cadherin phenotypes: mesotheliomas were positive for N-cadherin, and lung adenocarcinomas were positive for E-cadherin. Using antigen-retrieval methods, we successfully extended our study to formalin-fixed, paraffin-embedded tissue sections. Tumors from 28 patients (14 originally diagnosed as mesotheliomas, and 14 diagnosed as adenocarcinomas) were stained with 13A9 MAb and E9 MAb. Review of hematoxylin-eosin sections excluded from analysis one case previously diagnosed as mesothelioma, which represented a hemangiopericytoma. Of the remaining 27 cases, 12 of 13 mesotheliomas were positive for N-cadherin and negative for E-cadherin. The exception was a multifocal microscopic papillary tumor of apparent mesothelial origin, which was negative for both N-cadherin and E-cadherin. Conversely, 13 of 14 adenocarcinomas were E-cadherin positive and N-cadherin negative except for one adenocarcinoma with focal N-cadherin expression. One case of a poorly differentiated adenocarcinoma invading skeletal muscle was negative for both 13A9 and E9. These studies confirmed the utility of the cadherin antibodies in distinguishing pleural mesotheliomas from lung adenocarcinomas. The reactivity of the cadherin-specific antibodies with antigens in paraffin sections make them powerful and reliable markers in the practice of diagnostic surgical pathology.

Expression of E-cadherin and N-cadherin in surface epithelial-stromal tumors of the ovary distinguishes mucinous from serous and endometrioid tumors

This study examines the expression of E-cadherin and N-cadherin in the most common epithelial tumors of the ovary. The homotypic interactions of distinctive members of the cadherin family of cell-cell adhesion molecules segregate cells into tissues during embryonic development, and their expression in tumors can be used to trace the histogenesis of tumor cells. Because the surface epithelium of the ovary is a modified mesothelium, we speculated that the expression of E (epithelial)-cadherin and N (neural, mesodermal) -cadherin may provide clues about the controversial origin of common epithelial ovarian tumors. Immunohistochemistry was performed in paraffin sections using well-characterized monoclonal antibodies to E- and N-cadherin and heat-induced antigen-retrieval methods. We found that serous and endometrioid tumors express both E- and N-cadherin. In contrast, mucinous tumors strongly express E-cadherin, but no N-cadherin. The presence of N-cadherin in serous and endometrioid tumors traces their origin to the mesoderm-derived ovarian surface epithelium. The absence of N-cadherin in mucinous tumors clearly distinguishes them from the former, suggesting histogenesis from a cell lineage other than the ovarian surface epithelium or aberrant differentiation mechanisms associated with neoplastic transformation.

An optimal antigen retrieval method suitable for different antibodies on human brain tissue stored for several years in formaldehyde fixative

In the last 5 years the microwave oven has become a widely accepted apparatus for retrieving all kinds of antigens that are masked by prolonged formaldehyde fixation. However, it has so far not been possible to obtain good results for all the antigens we were interested in with just one method. This study offers a solution for this problem. We tested Tris buffered saline (TBS) pH 8–10 in order to see whether a microwave (MW) pretreatment in a solution with a high pH works on the antigens that have our particular interest: MAP-2, non-phosphorylated part of the neurofilament (SMI-32, SMI-311), phosphorylated part of the neurofilament (SMI-312) and the calcium binding proteins Calbindin D28-K, Parvalbumin and Calretinin. Furthermore we checked whether or not a lower temperature of 90°C (instead of boiling the tissue) could be used to achieve the same good staining results. These tests showed that an MW pretreatment in TBS pH 9.0 at full power (boiling) gave the best results. Pretreatment at a lower temperature gave also good results for most (but not all) antibodies when the irradiation time was prolonged to 30 min. The optimal antigen retrieval method also improved the quality of staining with an antibody against neuropeptide Y (NPY), for which, until now, no antigen retrieval appeared to be necessary, even after a long duration of fixation.