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PubMed1990

Immunohistochemical identification of infectious pancreatic necrosis virus in paraffin-embedded tissues of Atlantic salmon (Salmo salar).

Infectious pancreatic necrosis virus serotype Sp was identified by immunohistochemistry in formaldehyde-fixed and paraffin-embedded tissue of Atlantic salmon (Salmo salar). The immunoreaction was present in degenerating and necrotic cells in exocrine pancreatic cells. Cross reactions were observed with rabbit antisera against serotypes Sp, Ab, and VR-299 in neutralization tests and western blotting. Immunohistochemically, only Sp antiserum produced positive immunostaining to Sp antigens, whereas antisera to serotypes Ab and VR-299 were negative.

Animals↗
PubMed1981

Herpes simplex encephalitis. A modified indirect immunoperoxidase technique for rapid diagnosis in paraffin-embedded tissue.

A modification of a reliable and simple method of the indirect immunoperoxidase technique was used to identify herpesvirus antigen in paraffin-embedded tissue of patients with herpes simplex encephalitis (HSE), as well as in rats and mice. So far we have studied five cases in humans and six in mice and rats. With preincubation, we increased antigenicity and accomplished high specificity. Brown reaction product was found mainly at the periphery of the areas of necrotizing encephalitis. Here it was present in different types of glial cells, as well as in neurons. In the animals, it was found in the leptomeninx in cells of the basal cortex and in the ependymal cells of the third ventricle and the cells around it. This method should be of great value for the study of human pathology because new drugs are available for treatment of HSE and a tissue diagnosis is necessary. It is also a useful technique for studying viral CNS infections in experimental animals.

Animals↗
PubMedDate not supplied

Use of formalin fixed, paraffin embedded tumours for estimation of cellular DNA content and S-phase fraction by static cytofluorometry.

Sections with a thickness of 50 micron from 34 formalin fixed, and paraffin embedded tumours were deparaffinized and hydrated and the cells were disaggregated with pepsin. The cells were stained with Hoechst 33258 and a rapid method for static fluorometry was used. DNA ploidy and the fraction of cells in S-phase were estimated and compared with results obtained with freshly prepared cells. The coefficients of variation for the tumour stemlines for both the fresh and the embedded material were approximately 6 per cent. There was a good correlation between DNA ploidy determined from fresh and embedded material, respectively; there was also a close correlation between the fractions of S-phase cells estimated from fresh and embedded tumours. It was also possible to cut out small histologically defined lesions, such as carcinoma in situ of the breast from thick paraffin sections and obtain DNA histograms with high resolution.

Bisbenzimidazole↗
PubMed1982

Detection of anti-rotavirus antibody-producing cells in paraffin-embedded tissue sections.

An assay to detect anti-rotavirus antibody-producing cells was developed in rabbits. Sections of alcohol-fixed, paraffin-embedded tissues were used to make an immunofluorescent "sandwich" consisting of specific antibody-producing cells, a concentrated suspension of bovine rotavirus, and rhodamine-conjugated anti-rotavirus serum. Rhodamine-stained, anti-rotavirus antibody-producing cells could be simultaneously stained by a fluorescein-conjugated antiserum reactive to rabbit immunoglobulin.

Animals↗
PubMed1989

LN-6: a monoclonal antibody to vimentin expressed in non-hematopoietic mesenchymal cells and derived tumors and reactive in B5-fixed, paraffin-embedded tissues.

We generated a monoclonal antibody (MAb), designated LN-6, directed against human vimentin, which retains its immunoreactivity in B5-fixed, paraffin-embedded tissues. Like other anti-vimentin MAb, LN-6 was found to be reactive with a wide spectrum of human sarcomas and normal cells of mesenchymal derivation. However, unlike other similar reagents, LN-6 was unreactive with normal and malignant human lymphoid cells and therefore displays a more restricted immunoreactivity. Because of its ability to stain routinely processed pathological tissues and its marked reactivity with human sarcomas, LN-6 is a unique reagent for the immunohistochemical diagnosis of human cancer.

Animals↗
PubMed1985

Quantitative analysis of collagen, protein and DNA in fixed, paraffin-embedded and sectioned tissue.

Modifications and adaptations of chemical techniques have been used to quantitate hydroxyproline (collagen), total protein and DNA in fixed, paraffin-embedded and sectioned tissue. The assays are rapid and sensitive to between 0.1 and 1.0 micrograms of each of the three components. Values for the ratio of collagen to total protein or collagen per DNA obtained from sectioned material are the same as values derived from fresh or fixed starting material. Depending on the tissue and the sample size, as little as three to five 5 micron thick sections can be analysed for these three components. The ratios of collagen per total protein or collagen per DNA are given for several murine tissues. The assay of these components in tissue sections will allow a more precise correlation between histological appearance and the quantitative biochemical changes that may accompany many pathological disorders. The techniques are particularly useful in retrospective studies where the experimental material may be en bloc.

Animals↗
PubMed2002

DNA genotyping of unbuffered formalin fixed paraffin embedded tissues.

Formalin-induced DNA degradation was studied at different fixation times (3, 7, 16 and 32 days) each on 10 formalin fixed paraffin embedded tissues (FFPET) stored for 15 years at room temperature. The four different extraction protocols used in this study showed that Chelex100 extracts performed the best at 3 and 7 days of formalin fixation (DFF) (with regard to the quantity and the quality of the DNA). However, Qiamp extracts showed better results for long sized alleles, as well for single polymerase chain reaction (PCR) amplifications after 16 and 32 DFF, as for multiplex PCR at shorter fixation times. DNA degradation is expressed by the size of the amplified alleles, only 100 bp templates surviving after 32 DFF (AMG locus). Single locus amplifications (CD4 and FES/FPS alleles) performed better than multiplex PCR (ProfilerPlus), with nearly 100% positive results at 7 DFF. In both types of amplifications, the success rate decreased proportionally with the time of formalin fixation and, consequently, with the size of the required DNA template.

Alleles↗
PubMed1994

A simple, rapid method for isolating RNA from paraffin-embedded tissues for reverse transcription-polymerase chain reaction (RT-PCR).

Described here is a simple, rapid technique for isolating RNA from archived formalin-fixed, paraffin-embedded tissues (PETs). Using this modified acid guanidinium thiocyanate method, RNA sufficient as a template for the reverse transcription-polymerase chain reaction (RT-PCR) can be isolated in 2 hr from a single 20-microns-thick section of tissue. Spliced mRNA corresponding to portions of the estrogen receptor (ER) gene was successfully amplified from fixed, embedded human breast cancers containing increased amounts of ER protein. This method makes it feasible to safely and efficiently isolate RNA from large numbers of routinely processed paraffin blocks for retrospective studies of endogenous proteins such as hormone receptors, oncogenes, and viruses.

Base Sequence↗
PubMed1995

Patterns of immunoreactivity to an anti-fibronectin polyclonal antibody in formalin-fixed, paraffin-embedded oral tissues are dependent on methods of antigen retrieval.

The influence of antigen retrieval (AR) technique on immunoreactivity in formalin-fixed, paraffin-embedded tissues is recognized. In focal reactive overgrowths of oral mucosa, we noted that patterns of immunoreactivity to a fibronectin polyclonal antibody were dependent on methods of AR. To establish these patterns we investigated eight pyogenic granulomas and eight fibroepithelial polyps. In the absence of AR no immunoreactivity was observed. After alpha-chymotrypsin AR a band of intense immunoreactivity was associated with vascular endothelial cells, with either minimal or no staining of connective tissue. After microwave AR immunoreactivity was observed in connective tissue, especially in the subepithelial region, but there was no specific vascular staining. After autoclave AR immunoreactivity was observed in connective tissue and also in epithelial nuclei. To determine if these results represented either changes induced by tissue fixation and processing or exposure of epitopes normally masked in vivo, we investigated frozen sections from two fibroepithelial polyps and one pyogenic granuloma. In frozen sections immunoreactivity was observed around vascular endothelial cells and within connective tissue, especially in the subepithelial region, but not in epithelia. This study provides indirect evidence that alpha-chymotrypsin and heat-mediated AR protocols expose different masked epitopes, and reinforces the need to undertake appropriate pilot studies for immunohistochemical investigation of archival tissue.

Antibodies↗
PubMed1991

[Flow cytometric analysis of DNA content in paraffin-embedded tissue in head and neck cancer--evaluation of malignant potential and carcinogenic process of nasoparanasal tumor].

Flow cytometric analysis of DNA content from paraffin-embedded material has become an important diagnostic and prognostic method in clinical pathology and investigative oncology. We analyzed nuclear DNA content in order to detect possible alternations in DNA histogram as an indicator of malignant potential and carcinogenic process of head and neck tumor. DNA histograms were evaluated by three parameters; DNA aneuploidy, S + G2M% : rate of S and G2 + M phase cells as a parameter for growth kinetics, and polyploid%: rate of more than tetraploid cells as a parameter of nuclear atypia. We took a simple method for the selection of tumor area in paraffin blocks using a consecutive section stained with hematoxylin-eosin as a diagnostic guideline. This technique can be used either to enrich the sample to be analyzed with aimed area or to analyze histopathologically different compartments of the tumor. We compared the result of fresh and fixed specimens in 20 materials. DNA aneuploidy was found in both specimens of the same two carcinomas and there was a close relationship between them in S + G2M% (p less than 0.01) and polyploid% (p less than 0.05). We studied two cases of maxillary carcinoma with coexisting inverted papilloma as precancerous lesion. In one case S + G2M% and polyploid% were 16%, 1.15% in nasoparanasal papilloma, 20.5%, 4.0% in papilloma with atypia, and 33%, 8.25% in carcinoma, respectively. In the other case those were 3%, 0.1% in transitional papilloma, 8%, 0.9% in inverted papilloma, and 13%, 4.0% in carcinoma, respectively. There was positive correlation between these two parameters and histopathological grade. Finally we analyzed nuclear DNA content from fixed specimens in three groups; 33 papillomas, 15 maxillary carcinomas and 23 normal epithelia. Mean values of S + G2M% and polyploid% were as follows: 2.1%, 2.6% in normal epithelia, 19.6%, 8.9% in papillomas, and 34.8%, 18.1% in carcinomas. There was statistical significance between three groups (p less than 0.01). DNA aneuploidy was only found in 6 of 15 carcinomas (40%). The results demonstrated that S + G2M% and polyploid% were significantly compared with the histopathological grade of atypia and DNA aneuploidy was a marker of carcinoma. We suggest that DNA histogram is a good indicator for biological activity and that the increased S + G2M% and polyploid% may indicate carcinogenic process. We also suggest that tumor progression may lead to acquired genetic variability and DNA aneuploidy.

Aged↗
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↗
PubMed1984

Myelin basic protein in the optic nerve of the developing albino rat: an immunoperoxidase study of paraffin embedded tissue.

The distribution of myelin basic protein (MBP) was determined by application of the unlabeled peroxidase-anti-peroxidase method to sections of paraffin embedded optic nerve taken from the developing albino rat. MBP was not detected in optic nerves from animals younger than 90 hours postnatum. MBP staining presented first as faint, diffuse deposits of chromagen which were found in approximately one-third of the 90 hour subjects. The number of MBP positive regions and the intensity of staining of these regions increased rapidly with age. There was no obvious radial gradient in the distribution of MBP in cross sections of developing optic nerves, nor were smooth central/peripheral gradients apparent in longitudinal sections.

Age Factors↗
PubMed1980

Protein A-peroxidase conjugates for two-stage immunoenzyme staining of intracellular antigens in paraffin-embedded tissues.

Staphylococcal protein A conjugated to horseradish peroxidase was employed in an indirect immuno-staining technique to identify intracellular antigens in paraffin-embedded tissues. The sections were incubated with specific antisera and the antigen-IgG complexes demonstrated with protein A-peroxidase conjugate. Immunoglobulins, lysozyme and insulin were satisfactorily detected by this technique. A comparison of this method with the PAP, "labelled antigen" and peroxidase-labelled antibody sandwich techniques was made.

Antigens↗
PubMed1992

The cellular infiltrate in the liver of patients with fulminant hepatitis: analysis of paraffin-embedded tissue sections.

Intrahepatic infiltrate from 18 patients who died of fulminant hepatitis, was analyzed by an immunohistochemical method using formalin-fixed, paraffin-embedded liver sections and monoclonal antibodies. Inflammatory cells were characteristically located in the portal and periportal areas adjoining resting hepatocytes, but were infrequently found in the perivenular areas where hemorrhagic hepatocyte necrosis predominated. In the inflammatory infiltrate, T cells were the most predominant cell type, composing about two-thirds of the total hepatic infiltrate, followed by lysozyme-positive macrophages which composed about one-third of the total hepatic infiltrate, irrespective of the etiology of the fulminant hepatitis. On the other hand, B cells made up less than 2% in all cases, and plasma cells were also few, less than 2% in 12 of 18 cases. Furthermore, an enhanced display of beta 2-microglobulin on hepatocyte membranes was demonstrated in all cases with remaining hepatocytes, indicating an increased expression of class I MHC antigens on these cells. These results suggest that T cells may play an important role in the pathogenesis of the portal and periportal lesions of fulminant hepatitis, probably with a help of MHC class I antigens on hepatocytes, while hemorrhagic necrosis of hepatocytes around the central veins may be caused by a different mechanism, most likely a circulatory disturbance secondary to cell-mediated immune reactions in the periportal areas.

Acute Disease↗
PubMed1984

[Immunologic diagnosis of cutaneous lymphomas. I. A new immunoperoxidase technic for the demonstration of cell- and tissue-bound antigens in formalin-fixed paraffin-embedded tissues using protein A-peroxidase as a universal second antibody for mono- and polyclonal antibodies].

Immunoperoxidase techniques, applied to formalin-fixed paraffin embedded tissues, provide a simultaneous histopathological and immunological diagnosis. Application of protein-A-peroxidase constitutes a new technical variant: The technique is relatively simple, decreases non-specific background staining, and binds mono- and polyclonal antibodies from different mammalian species.

B-Lymphocytes↗
PubMed1993

Feline leukemia virus detection by immunohistochemistry and polymerase chain reaction in formalin-fixed, paraffin-embedded tumor tissue from cats with lymphosarcoma.

The prevalence of feline leukemia virus (FeLV) antigen and DNA was assessed in formalin-fixed, paraffin-embedded tumor tissues from 70 cats with lymphosarcoma (LSA). Tissue sections were tested for FeLV gp70 antigen using avidinbiotin complex (ABC) immunohistochemistry (IHC); DNA was extracted and purified from the same tissue blocks for polymerase chain reaction (PCR) amplification of a 166 base pair region of the FeLV long terminal repeat (LTR). Results were related to antemortem FeLV enzyme-linked immunosorbent assay (ELISA) for serum p27 antigen, anatomic site of LSA, and patient age. Viral DNA was detected by PCR in 80% of cases and viral antigen by IHC in 57% of cases. Seventeen cases were PCR-positive and IHC-negative; one case was PCR-negative and IHC-positive. Clinical records included FeLV ELISA results for 30 of 70 cats. All 19 ELISA-positive cats were positive by PCR and IHC; of the 11 ELISA-negative cats that were negative by IHC, seven were positive by PCR. When evaluated according to anatomic site, FeLV DNA and antigen were detected less frequently in intestinal LSAs than in multicentric and mediastinal tumors. Lymphosarcoma tissues from cats < 7 yr were several fold more likely to be positive for FeLV antigen by IHC than were tumors from cats > or = 7 yr. However, there was no significant difference in PCR detection of FeLV provirus between LSAs from cats < 7 yr and those > or = 7 yr.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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↗