Fractin immunostaining for the detection of apoptotic cells and apoptotic bodies in formalin-fixed and paraffin-embedded tissue.
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Actins↗
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A technique for simultaneous microangiographic and cytochemical investigation of resin-embedded tissues.
A method for simultaneous acquisition of information on cytochemical and microangiographic characteristics of tissue after perfusion with contrast medium was developed. The technique is based on the use of hydrosoluble resin as embedding material and is illustrated by the demonstration of gamma-glutamyltranspeptidase activity and of the microcirculation in rat liver during chemically induced hepatocarcinogenesis and experimental metastasis. The method is applicable to investigations aimed at correlating microangiographic and cytochemical characteristics of normal or altered tissues.
Angiography↗
Detection of human papillomavirus DNA in cell scrapes and formalin-fixed, paraffin-embedded tissue of the uterine cervix by filter in situ hybridisation.
Filter in situ hybridisation (FISH) was used to detect the presence of DNA of human papillomavirus (HPV) types 6/11 or 16/18 in cell scrapes (CYTOFISH) and formalin-fixed, paraffin-embedded biopsies (HISTOFISH) taken from the uterine cervices of 19 women. Paraffin tissue sections collected for HISTOFISH were either digested with pepsin or lysed with alkali/Triton X-100. The digest or lysate of the tissue sections and cell scrapes were applied to nylon or nitrocellulose membranes for nucleic acid hybridisation using 32P-labeled HPV-DNA probes. CYTOFISH and HISTOFISH were compared directly by taking samples for each method from the cervices of the same women. Of 19 women examined by colposcopy, cytology, and histology, eight were assessed as normal and 11 had evidence of a cervical disorder and/or the presence of HPV infection. Whereas no HPV-DNA was detected in the normal cases, the presence of HPV-DNAs was detected by both CYTOFISH and HISTOFISH in 11 cases with histological evidence of HPV infection and/or dysplasia. In these HPV positive cases, eight contained HPV 16/18, two HPV 6/11, and one a mixed infection of HPV 6/11/16/18. The high correlation between the results of CYTOFISH and HISTOFISH shows that formalin-fixed, paraffin-embedded cervical biopsies are suitable specimens for the detection and typing of HPV-DNA by FISH. Both CYTOFISH and HISTOFISH should facilitate studies on the prevalence and distribution of HPVs and their association with neoplasia.
Autoradiography↗
Immunophenotyping of acute leukemias using paraffin-embedded tissue sections.
In a study of 55 patients with either acute lymphoid leukemia (ALL; 25 cases) or acute myeloid leukemia (AML; 30 cases), paraffin-embedded bone marrow particle sections were examined with a panel of monoclonal and polyclonal antibodies reactive toward lymphoid and myeloid-associated antigens, using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. All cases were previously classified according to the French-American-British (FAB) Co-operative Group, and cases of ALL were immunophenotyped by flow cytometry. Results indicated that myeloid-associated antibodies (Mac 387, KP 1 [CD68], antielastase, antilactoferrin, and antilysozyme) did not react with any case of ALL, M1-AML, or M6-AML, whereas at least one of these antibodies reacted with 20 of 21 (95%) cases of M2, M3, M4, and M5-AML. Anti-glycophorin C marked cases of M6-AML, whereas anti-CD3 labeled T-cell ALL. None of the antibodies tested specifically identified cases of B-cell ALL. The authors conclude that use of a selected panel of antibodies on paraffin-embedded bone marrow particle sections may be of value in the diagnosis and immunophenotypic classification of many cases of acute leukemias.
Acute Disease↗
[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↗
Detection of human papillomavirus type 16 DNA sequences in paraffin-embedded tissues from the female urinary tract.
We investigated the presence of human papillomavirus-related DNA sequences (HPV 6, 11, 16 and 18) in 33 formalin-fixed paraffin-embedded biopsies from the urinary tract of female patients with recurrent and persistent urethritis and cystitis, using the polymerase chain reaction (PCR). The samples for PCR reaction were selected among tissues examined for histological diagnosis on the basis of the presence of microscopic changes consistent with HPV infection. Sequences homologous to HPV 6, 11 and 18 genome were not found, while HPV 16-related DNA sequences were identified in 25/33 lesions with histopathological diagnosis of metaplasia (1 from the urethra, 23 from the trigone and 1 from the bladder). The results suggest that the spread of HPV in the female urinary tract may not be uncommon and point to the need for further research on the possible pathogenic role in recurrent female disturbances.
Cystitis↗
Use of nanogold followed by silver enhancement and gold toning for preembedding immunolocalization in osmium-fixed, Epon-embedded tissues.
A reliable, sensitive, high-resolution method with good structural visualization for preembedding immunoelectron microscopy was proposed. The present technique involves the following processes. Immunolabeling of cryostat sections with primary antibodies and with nanogolds, silver intensification for visualization of nanogold secondary antibodies, gold-toning for stabilization of silver shells, and osmium postfixation and embedding in Epon for good structural visualization. The technique was applied to rat testis with anti-laminin and anti-fibronectin antibodies, showing that these two proteins are localized differentially in the lamina propria of the tissue.
Animals↗
METHOD OF SILVER IMPREGNATION FOR NERVOUS TISSUE EMBEDDED IN PARAFFIN.
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Coloring Agents↗
Staining method to demonstrate urate crystals in formalin-fixed, paraffin-embedded tissue sections.
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Birefringence↗
Bromodeoxyuridine (BrdUrd) immunohistochemistry in undecalcified plastic-embedded tissue. Elimination of the DNA denaturation step.
We examined the application of BrdUrd immunohistochemistry to detect S-phase cells in undecalcified bone and cartilage from the growing rat embedded in Spurr's resin. The effect of fixation on the procedure was studied, and the validity of the technique examined by a comparative study with tritiated thymidine ([3H]-TdR) autoradiography. The use of sodium-ethoxide to remove plastic from tissue sections prior to immunohistochemistry resulted in the production of sufficient ssDNA to make a separate DNA denaturation step unnecessary, thus sparing sections from potentially destructive treatment and shortening the immunohistochemical procedure. Fixation in formalin or Bouin's fluid gave the most satisfactory results. The distribution of BrdUrd labeled cells was restricted to the sites of cell proliferation in growing long bones. Combined studies with BrdUrd immunohistochemistry and [3H]-TdR autoradiography showed that the majority of BrdUrd labeled cells had also incorporated [3H]-TdR, thus attesting to the validity of the technique. This novel approach is suitable for the study of undecalcified hard tissues as well as soft tissues.
Animals↗
HSP47 is a useful marker for skin fibroblasts in formalin-fixed, paraffin-embedded tissue specimens.
AIM: This study was undertaken to assess whether heat-shock protein (HSP)47 is a useful cell marker for skin fibroblasts in formalin-fixed, paraffin-embedded skin specimens. BACKGROUND: HSP47, a 47-kDa HSP, is a collagen-specific molecular chaperone localized in the endoplasmic reticulum. HSP47 plays an essential role in collagen biosynthesis in skin fibroblasts. METHODS: Immunohistochemistry was performed to detect HSP47 in skin fibroblast cultures and skin tissue sections. RESULTS: Immunostaining for HSP47 clearly detected skin fibroblasts in paraffin tissue sections as well as in fibroblast cultures and frozen tissue sections. HSP47 staining on paraffin sections from diseased skin specimens revealed that skin ulcer, keloid, nodular fascitis, spindle cell lipoma, and dermatofibroma had strong signals for HSP47 compared with the signals obtained from normal skin. Dermatofibrosarcoma protuberans had many HSP47 positive cells, but signals on individual cells were not as strong as those seen from the above benign proliferative disease samples. In neurofibroma, a small number of faintly positive cells were detected. Our double-immunostaining studies also demonstrated that HSP47 staining distinguished skin fibroblasts from CD68-positive histiocytes/macrophages, factor VIII-related antigen-positive endothelial cells, or factor XIIIa-positive dermal dendritic cells. CD34-positive interstitial cells coexpressed HSP47 in spindle cell lipoma. CONCLUSIONS: These findings indicate that HSP47 staining can detect skin fibroblasts in routine, paraffin-embedded specimens. A panel approach using HSP47 and other cell markers on paraffin sections may help the identification of the cell type involved with mesenchymal proliferative disorders.
Biomarkers↗
Rapid detection of mycobacteria in inflammatory necrotizing granulomas from formalin-fixed, paraffin-embedded tissue by PCR in clinically high-risk patients with acid-fast stain and culture-negative tissue biopsies.
A collection of inflammatory necrotizing granulomas (INGs) negative by acid-fast stain and culture (AFSC) were analyzed by polymerase chain reaction (PCR) for the presence of mycobacteria. Forty-two paraffin-embedded specimens with INGs were collected from patients at high risk for contracting tuberculosis. Twenty biopsies were positive and 22 were negative for mycobacteria by AFSC. Two universal primers specific for all mycobacteria were used to detected a 414 base pair (bp) fragment of 16S rRNA gene. Twenty of 20 biopsies were positive for mycobacteria by both AFSC and PCR (100%), whereas 19 of 22 biopsies negative by AFSC were positive by PCR (86%). Follow-up of patients who were PCR positive but AFSC negative identified nine patients who had subsequent biopsies. Specimens from eight of these nine patients eventually grew Mycobacterium tuberculosis. Our results demonstrate that the detection of mycobacterial DNA by this method should be used in conjunction with AFSC for the initial diagnosis of mycobacterial infection.
Adolescent↗
Identification of cell wall deficient forms of M. avium subsp. paratuberculosis in paraffin embedded tissues from animals with Johne's disease by in situ hybridization.
M. avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease (JD) in ruminants leading to enormous economical losses in dairy and meat industries worldwide. During the subclinical stage of the disease, the infected animals are difficult if not impossible to detect by the available diagnostic tests including the PCR based ones. Although only considered an animal pathogen, cell wall deficient (CWD) forms of M. paratuberculosis have been isolated from patients with sarcoidosis and Crohn's disease (idiopathic diseases) in humans. Hence, the CWD form of this organism has been suspected to play a role in the pathogenesis of these diseases by persisting in the affected tissues and triggering a localized immune response and pathology. Differentiating between the CWD and acid-fast forms of this organism may lead to the determination of whether the CWD form is the pathogenic form in the subclinical cases of JD in animals and/or the etiologic agent for the above human diseases. To localize such organisms in tissue sections, CWD forms of mycobacteria were prepared in vitro and injected into beef cubes which were then formalin fixed and paraffin embedded. An in situ hybridization (ISH) technique, combined with the IS900 M. paratuberculosis-specific probe labeled with digoxigenin, was developed for the detection of nucleic acids specifically from the CWD forms but not their acid-fast forms in tissue sections. Specificity was confirmed by the negative finding with an irrelevant probe and with control tissue preparations containing CWD cells of related mycobacteria and unrelated organisms. This ISH procedure provides a way to distinguish between the acid-fast and CWD forms of M. paratuberculosis and to localize them in tissue sections. ISH may prove useful to evaluate the significance of CWD forms of M. paratuberculosis in the pathogenesis of JD, Crohn's disease and sarcoidosis.
Animals↗
Comparison of fresh and paraffin-embedded tissue as starting material for DNA flow cytometry and evaluation of intratumor heterogeneity.
Flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) from paraffin-embedded tumors has become an important diagnostic and prognostic tool in clinical pathology and investigative oncology. The present study aimed at elucidating the reliability of the method. About 90% of the 1,400 paraffin-embedded tumors analyzed were evaluable for DNA index and about 70% for SPF, although considerable differences between various tumor types were detected. The within-assay coefficients of variation for determination of tumor DNA index and SPF were 2% and 6%, respectively. Intratumor variation in DNA index was observed in 24% of breast and in 21% of ovarian carcinomas and variation in SPF in 36% and 29% of the respective tumors. Intratumor variation in SPF was greatest in DNA-diploid tumors, which may indicate that SPF values in these tumors may be less reliable owing to variations in the proportions of tumor and nontumor cells. If the methodological variation and the intratumor variation were taken into account, there was a good correlation between DNA indices (r = 0.980) and between SPF values (r = 0.794) obtained from fresh and paraffin-embedded tumors. It is concluded that accurate determination of DNA index and SPF from paraffin-embedded tumors is possible in the majority of cases. Regardless of the type of starting material used for DNA flow cytometry it is advantageous to study several samples from each tumor to account for the intratumor heterogeneity.
Breast Neoplasms↗
A method for the use of immunofluorescence on paraffin-embedded tissues.
Immunofluorescence is increasingly used for the visualization of antigens and antibodies in various tissues. The commonly used frozen sections present several disadvantages, which can be avoided by the present adaptation of paraffin-embedded sections for immunofluorescence. In this method, Bouin's solution achieves fast, deep fixation which preserves well the capacity of antigens to react with specific FITC-labeled antibodies. The method gives superior resolution of morphology, lower nonspecific fluorescent background, and permanent sections suitable for indefinite storage and retrospective studies. It has been successfully applied thus far to the study of immunofluorescence of lymphoid, thyroid, renal, pulmonary, and intestinal tissues.
Fluorescent Antibody Technique↗
[Detection of clonal immunoglobulin rearrangements in paraffin embedded tissues by PCR].
A polymerase chain reaction based method was established using immunoglobulin VH and VK gene rearrangements as markers to detect clonal B cell populations in paraffin embedded sections. Family specific VH and VK primers are used in separate reactions together with the corresponding J primers to amplify rearranged VH and VK genes from genomic DNA. This allows to distinguish clonal from polyclonal B cell populations in most of the cases. The method may be helpful in routine diagnosis of B cell NHL and some morphological and immunohistochemical difficult cases.
Base Sequence↗
Retrospective demonstration of endogenous peroxidase activity in plastic-embedded tissues conventionally prepared for electron microscopy.
A procedure is described that permits retrospective demonstration of intracellular endogenous peroxidase activity in tissue conventionally prepared for electron microscopy, i.e., doubly fixed with aldehydes and osmium tetroxide, "stained" in block with uranyl acetate, and embedded in epoxy resins. Using sodium ethoxide, plastic was removed from 1 micrometer sections; subsequently, the sections were incubated for 20 min in diaminobenzidine solution (44 mg/100 ml) made in acetate-citric acid buffer, pH 5.6, with 0.01% hydrogen peroxide. After this treatment, the sections were rinsed, dehydrated, and mounted. Cell types known to have endogenous peroxidase activity (red blood cells, macrophages, and retinal pigment epithelium cells in our preparations) show positive granules in their cytoplasm--control sections were uniformly negative. This method, which could also be used prospectively, cytochemically demonstrates endogenous peroxidase activity upon optical microscopical examination of the treated tissues; correlative electron microscopic studies may be performed on the same tissue block, or even adjacent sections.
Acute Disease↗
LacZ staining in paraffin-embedded tissue sections.
Femora and tibiae of rats carrying leukemia from a LacZ-marked acute promyelocytic leukemia-derived leukemic cell line (LT12NL15) were decalcified using EDTA and routinely embedded in paraffin. Sections were used to develop for the first time an immunostaining method for LacZ, employing catalyzed reporter deposition (CARD) based on the deposition of biotinylated tyramine. This method was used to study homing and adhesion of leukemic cells.
Animals↗