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PubMed1995

Immunocytochemical detection of androgen receptor in human temporal cortex characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues.

Immunocytochemical and biochemical studies have demonstrated the presence of androgen receptor protein in various regions of the rodent and non-human primate cortex. Localization of androgen receptor in the human brain has, however, not been studied as extensively, because of difficulties in obtaining suitable tissue samples. In the present study, we have localized androgen receptors in both frozen and paraffin-embedded temporal cortex from epileptic patients undergoing resection. Polyclonal antibodies were raised against fusion proteins containing fragments of the human androgen receptor protein. The antibodies were affinity-purified against the corresponding fusion protein. Immunoprecipitation and Western blotting using extracts from human cell lines demonstrated the specificity of the antibodies for the human androgen receptor and lack of cross-reactivity with other steroid hormone receptors. Immunocytochemistry was performed on frozen and paraffin sections of human temporal cortex and in paraffin-embedded benign hyperplastic prostates (BPH), as well as prostate and breast carcinomas, by the streptavidin-biotin-peroxidase method. Antigen-retrieval was performed in paraffin-embedded sections using microwave irradiation. Specific nuclear and cytoplasmic immunoreactivity for androgen receptor was detected in neurons, astrocytes, oligodendrocytes, and microglia cells of the temporal cortex. In contrast, only nuclear staining was observed in BPH, prostate and breast carcinomas. Immunoprecipitation of human temporal cortex lysate and subsequent Western blot analysis demonstrated the expression of a 98 kDa immunoreactive protein, slightly smaller than the reported molecular weight of the wild-type androgen receptor. These results provide further evidence for the expression of androgen receptor in the human temporal cortex. The use of these immunocytochemical techniques should enable the retrospective determination of possible changes in androgen receptor expression in a variety of archival paraffin-embedded tissues, including samples of the human central nervous system.

Adolescent↗

PubMed2004

Immunoperoxidase staining for C4d on paraffin-embedded tissue in cardiac allograft endomyocardial biopsies: comparison to frozen tissue immunofluorescence.

C4d deposition in microvasculature is a marker for humoral rejection. The authors compared a recently developed C4d immunoperoxidase (IP) method for paraffin-embedded tissue to immunofluorescence (IF) of frozen tissue. Of 315 frozen endomyocardial biopsies with IF staining for C4d, 280 were negative and 35 were positive. Negative controls were 17 negative biopsies and 11 biopsies with myocyte necrosis. The extent of IP and IF staining was graded as 0 to 3+. Staining intensity and the number and type of positive vessels were recorded. Staining patterns in Quilty lesions (QL) and foci of acute cellular rejection (ACR) were also evaluated. In 34 biopsies with sufficient tissue, IP criteria of 2+/3+, or more than 10 to 20 positive vessels per 10 high-power fields detected 25.0% (1/4), 18.2% (2/11), and 84.2% (16/19) of 1+, 2+, and 3+ IF-positive biopsies, respectively, without false positives. Considering C4d IF 3+ as positive resulted in 84.2% (16/19) sensitivity and 93.0% specificity (40/43). Intensely stained capillaries predominated in six of seven biopsies when more than 100 capillaries per 10 high-power fields were positive. Seventy percent (7/10) of IP 2+ and 3+ biopsies showed positive capillaries in QLs, while 36.4% (4/11) of IP 1+ and negative biopsies did. All eight IP 2+/3+ biopsies showed positive capillaries in ACR foci, while 25.0% (1/4) of IP-negative biopsies did. Capillary staining in QLs and areas of ACR reflects overall C4d deposition. In conclusion, IP staining of 2+/3+ is highly sensitive and specific for C4d positivity. The authors recommend considering 2+ and 3+ as positive staining when using the IP technique.

Adolescent↗

PubMed2003

Cross-reactivity of human and bovine antibodies in striped dolphin paraffin wax-embedded tissues.

This study evaluates the cross-reactivity of seven anti-human and one anti-bovine antibodies in formalin-fixed, paraffin-embedded tissue samples of liver and mesenteric lymph nodes of 13 striped dolphins (Stenella coeruleoalba). Four antibodies (CD3, IgG, lysozyme and S100 protein) reacted with striped dolphin lymph nodes in a similar pattern to that observed in the species of origin. The anti-human MHC class II mAb reacted strongly with macrophages and dendritic-like cells of striped dolphins, whereas a small number of lymphocytes were labelled with this antibody. These antibodies were used to study the immunophenotype of the inflammatory infiltrated in non-specific chronic reactive hepatitis (eight cases) and chronic parasite cholangitis (two cases) and normal liver (three cases) of striped dolphins. Non-specific chronic reactive hepatitis was composed of inflammatory infiltration of CD3+ T lymphocytes and IgG+ plasma cells in portal spaces and hepatic sinusoids. Lymphonodular aggregates observed in chronic parasitic cholangitis showed a cellular distribution similar to that found in lymph node cortex, including the presence of S100+ and MHC class II+ dendritic-like cells in lymphoid follicles and interfollicular areas. This result suggests that those inflammatory infiltrates are highly organised to enhance antigen presentation to B and T cells.

Animals↗

PubMed1991

A simple and rapid method for the detection of RNA in formalin-fixed, paraffin-embedded tissues by PCR amplification.

A simple and rapid method for the extraction, reverse transcription and PCR amplification of RNA from formalin-fixed, paraffin-embedded tissue is described. The procedure can be completed within 24 hours. In a first application of this method we detect human albumin mRNA in liver tissue, demonstrating the feasibility to retrospectively analyze gene expression and RNA viruses in fixed tissue.

Albumins↗

PubMed1991

Immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue sections. A comparison with genotypic analysis.

Several monoclonal antibodies (MoAbs) are now available for immunophenotyping non-Hodgkin's lymphomas (NHLs) in paraffin-embedded tissue sections. To determine the reliability of these reagents in predicting the genotype, 44 cases of NHL were studied with the alkaline phosphatase-anti-alkaline phosphatase technique with the use of the following MoAbs: leukocyte common antigen (CD45), Mac 387, L26, 4KB5, MB1, MB2, LN2, UCHL1, MT1, and MT2. The lineage of the neoplastic cells was determined in all cases by gene rearrangement studies for immunoglobulin heavy chain and for the T-cell receptor beta-chain. Genotypic results showed B-cell lineage in 33 cases (75%), T-cell lineage in 6 cases (14%), and mixed or undetermined lineage in 5 cases (11%). A concordance of lineage assignment by paraffin section immunophenotyping with gene rearrangement studies was observed in 37 of 39 (95%) lymphomas with an unequivocally defined genotype. MoAb L26 was the most sensitive in detecting B-cell genotype; MoAbs MT1 and UCHL1 were the most sensitive and specific, respectively, in detecting T-cell genotype. The authors conclude that lineage assignment of NHLs in paraffin sections is reflective of the corresponding genotype when an appropriate panel of MoAbs is used.

Antibodies, Monoclonal↗

PubMed1978

Identification of Rickettsia rickettsii in formalin-fixed, paraffin-embedded tissues by immunofluorescence.

With slight modification of a trypsin digestion technique, Rickettsia rickettsii were demonstrated specifically by immunofluorescence staining in Formalin-fixed, paraffin-embedded tissue sections from a human, rhesus monkey, and guinea pig with Rocky Mountain spotted fever and in infected membranes from a chicken embryo. Tissues were cut at 4 micron and, using geltain as a tissue adhesive, were hydrated in a routine manner. Sections were then digested in refrigerated 0.1% trypsin for 16 h, washed, and stained specifically for R. rickettsii by direct or indirect immunofluorescence. Rickettsial organisms were localized in affected vessels of the mammalian species and within the yolk sac epithelium of the chicken embryo. Specificity was confirmed by adsorbing antibody conjugates with R. rickettsii organisms. Trypsin digestion probably decreased tissue proteins which interfered with immunochemical attachment of antibody to the rickettsiae. The technique is valuable in that a diagnosis of Rocky Mountain spotted fever can be confirmed from Formalin-fixed tissues processed in a routine manner.

Animals↗

PubMed1993

Update on proliferation-associated antibodies applicable to formalin-fixed paraffin-embedded tissue and their clinical applications.

This review provides an update on proliferation-associated antibodies applicable to immunohistochemical techniques in formalin-fixed paraffin-embedded tissue. New insights into proliferating cell nuclear antigen (PCNA) and antibodies to PCNA are presented. The characterization of the protein recognized by the Ki-67 antibody has enabled production of a new range of antibodies (monoclonal and polyclonal) which have immunostaining profiles similar to those of the original antibody. A new proliferation-associated antibody, KiS1, is described. The clinical applications of antibodies to PCNA in human material are summarized, and the limitations of these studies are discussed.

Animals↗

PubMed1996

Immunoreactivity of new antibodies anti-p53 and anti-MDM-2 in paraffin embedded tissue samples.

Detection of various epitopes of the p53 and MDM-2 proteins, using new antibodies was performed on formalin-fixed and paraffin-embedded tissue samples from breast cancer and compared with results obtained using well-characterized antibodies. The results show that the distribution of positive nuclei and intensity of staining varies significantly depending on the antibody used, as well as on the microwaving procedure. Antibodies DO-14, DO-13 and SMP-14 have very good characteristics and are available for immunohistochemical analysis of p53 and MDM-2. Our results indicate, that immunohistochemical expression of p53 and MDM-2 is a not stable and unitary phenomenon and from this point of view a single antibody is not sufficient for its determination. Since, characteristics of p53 and MDM-2 molecules can vary from one sample to the next, panel antibodies capable of determining a wide range of wide type and mutant conformations must be used.

Antibodies↗

PubMed1998

A103: An anti-melan-a monoclonal antibody for the detection of malignant melanoma in paraffin-embedded tissues.

Melan-A is a previously defined, melanocyte differentiation antigen, and an anti-Melan-A murine monoclonal antibody, A103, was recently developed by our group. In this study, we evaluated A103 immunoreactivity on formalin-fixed, paraffin-embedded tissues, exploring the potential of A103 in the diagnosis of metastatic melanoma. Seventy-five metastatic melanomas, 10 primary melanomas, and 10 benign melanocytic nevi were tested. The reactivity of A103 was compared with HMB-4, an anti-gp100 antibody. Results showed that all nevi were A103 positive, and most primary melanomas were A103 and HMB45 positive. Of 75 metastatic melanomas, 61 (81%) were A103 positive, and 56 (75%) were HMB45 positive. Of 19 HMB45-negative lesions, 8 were A103 positive; of 14 A103-negative lesions, 3 were HMB45 positive. Eleven metastatic lesions, as well as 2 of 10 primary melanomas, were dual negative. These negative cases consisted mainly of the spindle cell and desmoplastic variants. Of the positive cases, A103 showed homogeneous staining in a significantly higher proportion of cases than HMB45 (72% versus 52%). In addition, focal staining with less than 5% reactive tumor cells was seen more frequently in HMB45 (12 of 56) than in A103 (5 of 61). These results indicated that A103 can be used as a first-line antibody in the diagnosis of metastatic melanoma. Our results also showed that A103 reacted with angiomyolipoma, which is known to be HMB45 positive. Of normal tissues, unexpected A103 reactivity was observed in the adrenal cortex, granulosa and theca cells of the ovary, and Leydig cells of the testis. This A103 immunoreactivity in benign and neoplastic tissues of nonmelanocytic origin, the basis of which is unclear, could also be of potential diagnostic value.

Angiomyolipoma↗

PubMed1991

An improved Hedley method for preparation of paraffin-embedded tissues for flow cytometric analysis of ploidy and S-phase.

A modification of the Hedley-method for flow cytometric DNA analysis of paraffin-embedded tissues is presented. Dewaxed and dehydrated tissue from paraffin blocks was incubated with subtilisin Carlsberg (pronase, Sigma protease XXIV) and then stained directly without washing and centrifugation. The loss of material was minimized, which was advantageous, for example, for the analysis of core-biopsies, and all measured samples showed extremely low frequencies of clumped cell nuclei. This made is easier to detect polyploid nuclei and even rare nuclei of high ploidy could be identified. S-phase analyses were more precise, since the background originating from clumped debris particles was very low. The improved method was applied to the estimation of frequencies of high-polyploid nuclei found in various diploid, tetraploid, and aneuploid human myosarcomas of the uterus.

Biopsy↗

PubMed1987

Detection of cytomegalovirus infection by means of DNA isolated from paraffin-embedded tissues and dot hybridization.

A specific and rapid method for detecting the presence of cytomegalovirus (CMV) DNA in formalin- or mercuric chloride-fixed, paraffin-embedded tissue has been developed. With dot hybridization with a biotinylated CMV DNA probe, the presence of CMV infection can be readily detected even if only a minority of the cells in the tissue block are infected. The sensitivity is further increased by about two orders of magnitude if high specific-activity 32P-labeled probe is used. This method of detection is more sensitive than histologic diagnosis, appears to be roughly comparable to that by virus isolation in culture, and should be applicable to the detection of other infectious agents.

Autopsy↗

PubMed1987

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA-binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1.

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

Animals↗

PubMed1994

Detection of human herpesvirus-6 in paraffin-embedded tissue of cervical cancer by polymerase chain reaction.

The polymerase chain reaction (PCR) was used to detect human herpesvirus-6 (HHV-6) DNA sequences from paraffin-embedded tissue from cervical cancer patients. Two of eight cases were positive for HHV-6 using two sets of HHV-6 primers. Hybridization of PCR products with specific radioisotope-labeled oligonucleotide probes confirmed the results. Furthermore, HHV-6 typing was possible by adapting restriction endonuclease digestion of PCR product. This method is useful for retrospective studies in investigating the etiologic role of HHV-6 in the development of human diseases.

Base Sequence↗

PubMed1988

Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections.

We show in this communication that polyacrylamide gel electrophoresis (PAGE) and immunoblotting of proteins can be performed using one to two 5-7 micron paraffin sections of tissues fixed in non-cross-linking fixatives (acetone, alcohol, or modified Carnoy's solution). Proteins for study were extracted from paraffin sections of mouse foot pad and liver. The presence of unaltered keratin polypeptides in tissues fixed with either acetone or alcohol was demonstrated in gels stained with Coomassie brilliant blue. The preservation of their antigenic determinants was demonstrated with immunoblotting. Furthermore, the immunoreactivity of soluble proteins, such as albumin, remained unaltered in immunoblots obtained from paraffin-embedded mouse liver sections. These data indicate that tissues embedded and stored in paraffin are useful for the above-mentioned biochemical and immunological studies and may therefore be an important technique for diagnostic pathology or retrospective studies.

Animals↗

PubMed1994

Detection of louping ill virus in formalin-fixed, paraffin wax-embedded tissues of mice, sheep and a pig by the avidin-biotin-complex immunoperoxidase technique.

An immunohistochemical method for the detection of louping ill virus antigen in formalin-fixed, paraffin wax-embedded tissues by an avidin-biotin-complex (ABC) immunoperoxidase technique was established. The tissues examined were from the brains of 10 mice, five sheep and one pig. The mice were experimentally infected with louping ill virus whereas the sheep and the pig were field cases of louping ill confirmed by clinical examination, and by histological and serological methods.

Animals↗

PubMed2001

Evaluation of extraction methods from paraffin wax embedded tissues for PCR amplification of human and viral DNA.

AIM: To evaluate the efficiency of phenol/chloroform, microwave, and Qiagen spin column based DNA extractions from paraffin wax embedded tissue for use in the polymerase chain reaction (PCR). In addition, to assess the reliability of amplifying a housekeeping gene to indicate successful viral DNA extraction. METHODS: DNA samples extracted from 20 blocks of cervical carcinoma tissues using the three methods were subjected to PCRs targeting 509 bp and 355 bp of the beta globin gene, and 450 bp and 150 bp of human papillomavirus (HPV) DNA. RESULTS: Microwave extraction showed the highest positive rate for beta globin PCR, whereas the spin column method was the most efficient for HPV DNA extraction. When the 509 bp beta globin and 450 bp HPV PCR results were correlated, two of 10, eight of 12, and nine of 10 beta globin positive extractions prepared by means of the phenol/chloroform, microwave, and spin column methods, respectively, yielded HPV DNA of the expected size. For the beta globin negative samples, HPV was detected in three of 10, two of eight, and four of 10 samples. CONCLUSIONS: HPV DNA extraction was most efficient using the Qiagen spin column and had the highest positive predictive value when a housekeeping gene was used as an indicator of successful viral DNA extraction; the phenol/chloroform method was the least efficient. The potential drawbacks of some extraction methods when using a human housekeeping gene to assess the quality of viral DNA extraction need to be considered.

Carcinoma, Squamous Cell↗

PubMed1986

Improved method for making high-affinity sections of soft tissue embedded in polyethylene glycol (PEG): its use in screening monoclonal antibodies.

This article describes improvements in the immunohistologic technique for embedding highly hydrated embryonic tissue in polyethylene glycol 1000 (PEG)--a water-soluble wax of melting point 39 degrees C--and compares the PEG sections with frozen and polyester-wax sections. The main improvement ensures that relatively large PEG sections (8 X 3 mm) stretch out and adhere well to slides: a coat of albumen and glycerine is dried onto the slides and a fresh coat applied just before use. The embedding, sectioning, and mounting procedures, which are considerably faster than those for wax processing, have been developed for screening monoclonal antibodies against the differentiated neural crest cells in the anterior eyes of 9-day-old chick embryos. PEG sections of such eyes were a little fragile, but showed good cellular detail, similar to or better than in wax sections and considerably better than in frozen sections. The responses of PEG sections to the antibodies were far stronger than those of wax and marginally better than those of frozen sections. In one experiment using 125I-labeled rabbit anti-mouse antibody on sections previously treated with antibodies or antisera, PEG sections bound about five times as much label as wax sections and approximately 30% more than frozen sections. The main limitation of the technique is that, because of the softness of PEG, it only works well for embedding a limited range of tissues. Such PEG sections may, however, be useful for in situ hybridization as well as for immunohistochemistry.

Animals↗

PubMed1991

PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time.

The polymerase chain reaction (PCR) DNA amplification method is a powerful new tool for the retrospective analysis of paraffin-embedded tissue (PET). The technique has afforded the sensitive and specific detection of nucleic acid sequences associated with genetic and infectious diseases. However, PET processing conditions vary in their suitability for amplification. The authors have examined the effects of 11 fixatives at three fixation times. The effect of fixation was measured by the ability of the DNA in a treated tissue to serve as a template for the amplification of DNA fragments that ranged from 110 to 1,327 base pairs in length. Specimens fixed in acetone or 10% buffered neutral formalin were found to be best suited for subsequent analysis by PCR. A second group of fixatives, including Zamboni's, Clarke's, paraformaldehyde, formalin-alcohol-acetic acid, and methacarn, compromised amplification efficiency. Tissues treated with Carnoy's, Zenker's, or Bouin's, respectively, were even less desirable for amplification analysis.

Acetone↗