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Results for “Paraffin Embedding”

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PubMed1988

Monoclonal antibodies marking B-cell non-Hodgkin's lymphoma in paraffin-embedded tissue.

Traditional methods for the immunophenotypic analysis of the non-Hodgkin's lymphomas require fresh or snap-frozen tissue for flow cytometric or immunohistochemical studies. The monoclonal antibodies LN1, LN2, and L26 have been recently developed to recognize B-cell-specific antigens that survive routine tissue processing and paraffin embedding. In this study, the ability of these three antibodies to mark the neoplastic cells in 160 cases of paraffin-embedded non-Hodgkin's lymphoma relative to frozen section immunophenotype (42 T-cell, 118 B-cell), manner of fixation (B5 versus 10% buffered formalin), and histological subtype was examined. With B5-fixed tissue, the percentages of B-cell lymphoma marking with the antibodies were as follows: L26, 96.6%; LN1, 88.2%; LN2, 93.7%. With formalin-fixed tissue, the percentages of B-cell lymphoma reacting with the antibodies were: L26, 89.1%; LN1, 26.2%; LN2, 57.8%. Each of the antibodies marked a small percentage of paraffin-embedded T-cell lymphomas: L26, 4.7%; LN1, 4.7%; LN2, 7.1%. LN2, and to a lesser extent LN1, stained Reed-Sternberg cells, a feature not seen with L26. Nor did L26 mark nonlymphoid neoplasms, a feature previously reported with LN1 and LN2. Since a high percentage of B-cell lymphomas react with these antibodies and they are relatively specific for B-cells, they should prove highly useful for the evaluation of both diagnostic and experimental pathology specimens. L26 offers the distinct advantage of working well in both B5 and formalin-fixed tissues and seemingly not marking epithelial neoplasms.

Antibodies, Monoclonal↗

PubMed2002

Genotyping of Helicobacter pylori in paraffin-embedded gastric biopsy specimens: relation to histological parameters and effects on therapy.

OBJECTIVES: Colonization with Helicobacterpylori can lead to GI disease. Bacterial genotypes and host factors, such as acid production, can influence the progress of disease. We investigated H. pylori genotypes and histological parameters in the same paraffin-embedded gastric biopsy specimens. METHODS: Paraffin-embedded antrum and corpus biopsy samples from 75 gastroesophageal reflux disease patients were histologically examined and tested for H. pylori vacA (s and m regions), cagA, and iceA genotypes. Patients were investigated at baseline (58 H. pylori positive and 17 H. pylori negative) and after treatment with omeprazole with or without additional antibiotic therapy. RESULTS: Genotyping at baseline was complete in 52 (90%) of the 58 H. pylori positive patients. Multiple genotypes were detected in eight (14%) of these. Genotypes were highly consistent between antrum and corpus biopsy specimens at baseline and in follow-up samples. Genotypes from paraffin sections matched those from corresponding cultured strains in 10 selected cases. In the antrum, the degree of inflammation was associated with vacA s1 and cagA+ genotypes, and the degree of neutrophil activity was associated with the cagA+ genotype. In the corpus, the degree of inflammation was significantly associated with vacA s1, cagA+, and iceA1 genotypes and the degree of atrophy was associated with vacA s1, m1, and cagA+ genotypes, whereas the degree of neutrophil activity was associated with vacA s1 and cagA+ genotypes. vacA s2 and cagA-strains appeared more resistant to antibiotic therapy, irrespective of resistance to clarithromycin. CONCLUSIONS: Our findings confirm the relevance of the H. pylori genotypes for the severity of gastric disease and the efficacy of antibiotic therapy.

Amoxicillin↗

PubMed1995

An improved method for the species-specific assessment of mycobacteria in routinely formalin-fixed and paraffin-embedded tissues.

A polymerase chain reaction (PCR) assay for the rapid and species-specific diagnosis of mycobacterial infections in paraffin-embedded clinical specimens was developed using oligonucleotide primers to amplify a fragment of the DNA coding for the ribosomal 16S RNA of mycobacteria. The oligonucleotide primers amplified DNA from all 14 species of mycobacteria tested. By means of a reamplification protocol, as few as one to two mycobacteria could be detected in the presence of human DNA. The method of DNA isolation and amplification was applied on sections of routinely formalin-fixed and paraffin-embedded tissues. PCR for the beta-actin gene served as a control for successful DNA isolation. Mycobacterial DNA could be detected in cases of mycobacterial infections. The mycobacterial species was determined by additional sequencing of the PCR fragment. This PCR method may be a powerful tool for the diagnosis of mycobacterial infections from histopathological material and for the assessment of those mycobacteria that cannot readily be cultured, such as Mycobacterium leprae.

Base Sequence↗

PubMed1986

Detection of Pi Z phenotype individuals by alpha-1-antitrypsin (AAT) immunohistochemistry in paraffin-embedded liver tissue specimens.

Immunostaining for alpha-1-antitrypsin (AAT) was applied on paraffin-embedded liver specimens from 38 Pi Z phenotype (27 Pi MZ; 9 Pi ZZ; 2 Pi SZ) individuals (33 adults, 5 newborns). Histological diagnoses included normal liver, various forms of chronic hepatitis, cirrhosis. A positive hepatocytic staining for AAT was revealed in all cases, whereas on PAS-D staining 5 cases were negative. Immunostaining revealed different patterns: type I: fine solid granules or coarse laminated globules filling the whole liver cell cytoplasm; type II: positive granules marginated towards the cell's periphery; type III: positive granules restricted to focal areas in the cytoplasm. Type I and III patterns were observed in all specimens. Association of type I and III with type II was seen in all Pi MZ and Pi SZ cases, but only in 3 of the 9 Pi ZZ specimens. To check the specificity of these staining patterns for the Z allele of AAT in adults and newborns, 180 further cases were investigated blindly by immunohistochemistry. Recognition of hepatocytic staining patterns type I, II and III allowed to reliably diagnose the Pi Z phenotype in 6 cases, whose heterozygous state was confirmed by independent serum phenotyping (5 Pi MZ, 1 Pi SZ). These results allow to conclude that: (1) immunohistochemistry is a reliable technique (superior to PAS-D staining) to identify Pi Z individuals (homo- and heterozygotes) on paraffin-embedded liver sections; (2) heterozygotes Pi MZ and Pi SZ cannot be differentiated without serum phenotyping; (3) immunohistochemistry is helpful in distinguishing between Z homozygotes from heterozygotes, but determination of AAT serum concentration is required to allow confident distinction without phenotyping. AAT immunohistochemistry thus allows to diagnose the Z allele of AAT when phenotyping procedures are not available or in retrospective studies on biopsy and autopsy material.

Adult↗

PubMed1991

Demonstration of oestrogen and progesterone receptors in freeze-dried, paraffin-embedded sections of breast cancer.

Immunohistochemical evaluation of oestrogen and progesterone receptors is of importance in evaluating human breast tumours. Staining techniques can be performed on snap-frozen, cryostat-cut tissues or, as recently reported, on formalin-fixed, paraffin-embedded tissues. These methods are, however, limited by several drawbacks, including difficulties in retrospective studies and in storage of the material, and the relatively high frequency of false negative results for chemically fixed specimens. We therefore investigated the application of freeze-drying technology to assess the feasibility and reliability of this technique as an alternative method for diagnostic breast pathology. Morphological and immunohistochemical studies were performed on snap-frozen, freeze-dried and paraffin-embedded tissue obtained from 16 cases of benign and malignant breast neoplasms. Our results showed good preservation of tissue morphology, similar to standard formalin fixation, and excellent preservation of antigenic reactivity of nuclear receptors, comparable to that obtained with cryostat sections. We therefore suggest that freeze drying and paraffin embedding of frozen tissue blocks is equivalent or even preferable to formalin fixation for the demonstration of oestrogen and progesterone receptors, at least in the case of small tumours.

Breast Neoplasms↗

PubMed2001

In situ hybridization and reverse transcription-polymerase chain reaction for cyclin D1 mRNA in the diagnosis of mantle cell lymphoma in paraffin-embedded tissues.

Mantle cell lymphoma (MCL) is characterized by the chromosomal translocation t(11;14), which involves rearrangement of the bcl-1 proto-oncogene to the immunoglobulin heavy chain gene and results in overexpression of cyclin D1 mRNA. In this study, we evaluated the diagnostic relevance of three methods that may be helpful in the diagnosis of MCL: in situ hybridization (ISH) and a stringent reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for cyclin D1 mRNA, and immunohistochemistry for cyclin D1 protein. The study group included 37 paraffin-embedded specimens (25 from lymph nodes and 12 from extranodal tissues) from 30 patients. MCL diagnosis was performed according to the Revised European-American Classification of Lymphoid Neoplasms. Twenty-nine patients with non-MCL lymphoproliferative disorders comprised the control group. Biotin-labeled ISH was performed in 28 cases of MCL, 24 (86%) of which were found to be positive. As shown by ISH in extranodal tissues, cyclin D1 mRNA was present not only in neoplastic lymphoid cells, but in other cell types as well. For this reason, RT-PCR results were considered reliable for MCL diagnosis only on informative material (from tissues that do not normally express cyclin D1); this method was evaluated as positive in 16 of 18 (89%) MCL cases. Cyclin D1 immunopositivity was present in 20 of 29 (69%) MCL cases. No members of the control group were found to express cyclin D1 mRNA by either ISH or RT-PCR under the stringent conditions used. In conclusion, stringent RT-PCR for cyclin D1 expression can be helpful in MCL diagnosis in paraffin-embedded material from lymph nodes. ISH is a sensitive method for cyclin D1 mRNA detection; its sensitivity is superior to that of cyclin D1 immunohistochemistry and similar to that of the stringent RT-PCR used. ISH is very specific as well, clearly more specific than RT-PCR, because it allows the correlation of molecular findings with morphology. This method can be applied on all types of paraffin-embedded tissues and provides an accurate tool for MCL diagnosis.

Cyclin D1↗

PubMed1996

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: an analysis of resected gastric carcinoma.

Although several factors affecting the sensitivity of polymerase chain reaction (PCR) amplification from formalin-fixed tissues have been investigated mostly by experiments, the feasibility of archival formalin-fixed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2-3 days and 14% (4/28) of the samples fixed for 4-6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified p53 products from archival tissues using a non-isotopic method, temperature gradient gel electrophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding just after 1 day or less fixation for subsequent use in PCR amplification.

Animals↗

PubMed2001

Diagnostic potential of fluorescence of formalin-fixed paraffin-embedded malignant melanoma and pigmented skin lesions: quantitative study of fluorescence intensity using fluorescence microscope and digital imaging.

The background for this study was reports in the literature of stronger fluorescence observed visually for melanomas compared with benign naevi in formalin-fixed paraffin-embedded sections. Our objective was to carry out a quantitative study of the phenomenon and to investigate if such an approach could be used in the detection of melanomas. Microscopic digital imaging was used to measure quantitatively the fluorescence intensity in specimens from 50 malignant melanomas, four basal cell carcinomas and 58 benign lesions. The mean fluorescence intensity of the melanomas was considerably higher than of the other lesions. For melanomas, the intensity depended both on the distance from the skin surface and the distance from the centre of the lesion. A simple algorithm based on the intensity threshold correctly classified the melanomas with a sensitivity of 74% and a specificity of 59%. Quantitative measurements of the fluorescence of the pigmented skin lesions fixed with formalin and embedded in paraffin can be a useful auxiliary tool for differentiating melanoma from other pigmented lesions histopathologically.

Basal Cell Carcinoma↗

PubMed1997

Immunohistochemical determination of hepatic cytochrome P-4502E1 in formalin-fixed, paraffin-embedded sections.

Cytochrome P-4502E1 (2E1) is inducible by chronic ethanol consumption that results in enhanced activation of anesthetics and commonly used drugs (such as acetaminophen) to hepatotoxins. Therefore, assessment of hepatic 2E1 is needed in prescribing these drugs for the management of alcoholic patients. Currently, measurement of 2E1 requires either immunohistochemistry on frozen sections or Western blot (WB) analysis of homogenized tissue in excess of that needed for pathology. To obtain a more widely applicable method, we developed a procedure to detect 2E1 by immunohistochemistry in formalin-fixed, paraffin-embedded liver biopsies obtained routinely for diagnosis. Data were collected from rats fed ethanol-containing or control liquid diets for 3 weeks. Immunostaining was performed using anti-human rabbit 2E1 antibody as the primary antibody, and the immunoreaction was detected by the avidin-biotin immunoperoxidase method after treating sections with target unmasking fluid, an antigen retrieval buffer that enhanced the staining of 2E1. In control rats, 2E1 staining was weak and perivenular. After ethanol feeding, it showed a lobular gradient, strongest perivenular and weakest periportal, similar to that seen in frozen sections. The staining intensity was scored as: 0 (no staining) to 3 (strong staining). The zonal staining was scored as follows: 1 = perivenular zonal staining, 2 = midzonal, and 3 = panlobular. With the product of the two scores, a significant difference was found between alcohol-fed and control rats (5.1 +/- 0.3 vs. 0.8 +/- 0.2, p < 0.001). 2E1 assessments by WB were also significantly different for these rat pairs (68.5 +/- 2.1 vs. 7.9 +/- 0.8 arbitrary units/mg protein, p < 0.001), with a parallel increase of immunostaining scores and WB measurement of 2E1 content. This immunohistochemical method was then validated in 14 paraffin-embedded percutaneous human liver biopsy samples. In livers of nonalcoholics, 2E1 staining was seen in the perivenular zone only, whereas in samples of alcoholics, the staining was perivenular to midzonal and sometimes periportal. A significant correlation between the zonal staining scores (rs = 0.67, p < 0.005) or intensity x zonal staining scores (rs = 0.79, p < 0.001) and WB analysis was found. The immunohistochemical assessments of 2E1 expression in formalin-fixed, paraffin-embadded sections from livers of alcoholics was found to correlate with WB analysis, and lobular distribution was consistent with that seen in frozen sections. The proposed method should therefore be useful for the assessment of 2E1 content in paraffin-embedded liver samples, thereby aiding in the management of heavy drinkers.

Adult↗

PubMed1992

Cytokeratin immunoreactivity in malignant fibrous histiocytoma and spindle cell tumors: comparison between frozen and paraffin-embedded tissues.

Cytokeratin (CK) immunoreactivity in malignant fibrous histiocytoma (MFH) and other selected cases of spindle cell tumors were assessed using two cytokeratin monoclonal antibodies, AE1/AE3 and CAM 5.2. Frozen tissue was used to minimize the effects of fixation on keratin antigenicity; in addition, one block of fixed, paraffin-embedded tissue was tested for comparison. CK immunoreactivity was noted in nine frozen tissue samples (7/20 [35%] MFH, 1/3 schwannomas, 1/3 leiomyosarcomas). In the majority of cases, only rare individual positive cells were seen. Of 19 MFH cases in paraffin-embedded tissue, CK immunoreactivity was noted in three (16%). All 32 cases examined showed vimentin immunoreactivity. MFH must be added to the growing list of mesenchymal tumors exhibiting sporadic CK immunoreactivity. Such reactivity is less frequent in paraffin-embedded tissues. This finding has important implications for tumor diagnosis, particularly in the differential diagnosis of pseudosarcomatous carcinoma. Caution is recommended in the interpretation of CK immunoreactivity, particularly as it relates to speculations regarding histogenesis.

Adult↗

PubMed1990

Analysis of antigen receptor gene rearrangements in ethanol and formaldehyde-fixed, paraffin-embedded specimens.

Molecular hybridization analysis of DNA prepared from frozen specimens obtained from patients with lymphoproliferative disorders has aided in the determination of the lineage and clonality of the neoplastic cells in many cases. We investigated whether high molecular weight DNA suitable for nucleic acid hybridization studies could also be prepared from fixed, paraffin-embedded material. After selecting nine representative cases, we extracted DNA from frozen sections by using standard methods, and from ethanol-fixed tissue in paraffin blocks. The yields of DNA from ethanol-fixed blocks were similar to yields from frozen tissue. DNA from frozen and ethanol-fixed tissues was subjected to Southern blot hybridization and probed for rearrangements of immunoglobulin heavy, and kappa, and lambda light chain genes, as well as for the T cell receptor beta-chain gene. In each of the cases, comparable results were obtained, regardless of the source of DNA. DNA extracted from ethanol-fixed blocks stored for 2 years gave identical results. We also prepared DNA from formaldehyde-fixed, paraffin-embedded tissue obtained from six of the patients. Five of the specimens yielded spoolable DNA (average recovered, 313 micrograms), but the DNA was degraded more than that obtained from the frozen or ethanol-fixed specimens. Formaldehyde-treated DNA gave variable results in Southern blot hybridization studies, and less than half of the results were interpretable. We conclude that ethanol-fixed, paraffin-embedded tissues provide an excellent source of DNA for nucleic-acid hybridization studies, and that they are easily handled and stored.

DNA↗

PubMedDate not supplied

A rapid and simplified technique for analysis of archival formalin-fixed, paraffin-embedded tissue by fluorescence in situ hybridization (FISH).

We here present a simplification of the entire procedure for preparing formalin-fixed, paraffin-embedded tissue to be used for FISH-analysis. The steps for deparaffinisation and disintegration of the tissue to produce intact cell nuclei in a monodispersed suspension are detailed as well as the hybridisation steps. The procedure results in a clear cell suspension with intact cell nuclei and without clumped debris particles. The enzymatic digestion is restricted to the trypsinisation step of the deparaffinisation procedure. No further pretreatments prior to hybridization are performed. This modified technique has been successfully applied to different formalin-fixed, paraffin-embedded materials.

Cell Nucleus↗

PubMed1995

Microwave antigen retrieval for immunocytochemistry on formalin-fixed, paraffin-embedded post-mortem CNS tissue.

Microwave antigen retrieval methods were assessed for a panel of antibodies on formalin-fixed, paraffin-embedded sections from a range of human central nervous system (CNS) tissues taken at post-mortem. The immunoreactivity of a number of antigens (synaptophysin, PGP9.5, GFAP, carbonic anhydrase II, CD68, ferritin, HLA-DR, alpha beta-crystallin, measles and herpes viruses) was markedly enhanced by this procedure compared with other methods of antigen unmasking, such as trypsinization. Enhancement was noted both by an increased number of positive cells and by the intensity of reactions within individual cells and their processes. Furthermore, the microwave method produced uniform immunostaining over large surface area sections with no loss of morphological detail. Large cryostat sections of CNS tissue can be difficult to obtain with good morphology. The ability to retrieve a wide range of antigen in formalin-fixed, paraffin-embedded CNS tissue will greatly increase the range of investigations that can be carried out on this type of stored material.

Antigens↗

PubMed1999

Detection of Mycobacterium avium subsp paratuberculosis in formalin-fixed paraffin-embedded intestinal tissue by IS900 polymerase chain reaction.

OBJECTIVES: To evaluate and compare methods for DNA extraction from formalin-fixed, paraffin-embedded tissues and methods for detection of Mycobacterium avium subsp paratuberculosis by IS900 PCR for confirmation of Johne's disease in ruminants. DESIGN: A laboratory study. PROCEDURE: Three methods of DNA extraction of differing complexity and two PCR protocols using different pairs of IS900 primers were compared. Sensitivity and specificity were assessed using samples from ruminants with and without histological evidence of Johne's disease. RESULTS: The simplest method of DNA extraction, which involved two cycles of boiling and freezing followed by centrifugation, gave more consistent results than two methods that required solvent extraction of paraffin, proteinase digestion and DNA purification. The sensitivity of detection of M avium subsp paratuberculosis in paraffin blocks stored for 1 to 6 years from 34 cases of Johne's disease in sheep, cattle and goats was 88% for a 229 bp IS900 PCR assay and 71% for a 413 bp assay, using the detection of acid-fast bacilli by Ziehl Neelsen staining of histological sections from the same blocks as the gold standard test. PCR results correlated with the abundance of acid-fast organisms in the tissues. No false positive reactions were detected. CONCLUSION: PCR for identification of M avium subsp paratuberculosis in formalin-fixed, paraffin-embedded intestinal tissues from ruminants is a rapid and useful method. A simple method of sample preparation is effective. Amplification of short fragments of IS900 is more effective than amplification of longer fragments.

Animals↗

PubMed2006

Tumor cell nuclei extraction from paraffin-embedded lymphoid tissue for fluorescence in situ hybridization.

In this study the authors evaluated a technique for isolating intact tumor nuclei from paraffin-embedded lymphoma samples before performing FISH testing to detect the lymphoma-specific trans-location t(11;14) that defines mantle cell lymphoma. Well-characterized surgical pathology cases of mantle cell lymphoma were identified from pathology archives. Thin sections were cut from the paraffin-embedded tissue blocks. One section was stained using hematoxylin and eosin and an area composed exclusively of malignant cells was identified and marked on the slide. The corresponding area of the tissue block corresponding to this region underwent needle core biopsy, and the tissue was processed to isolate tumor cell nuclei and deposited onto a glass slide. The paired sample preparations underwent routine FISH testing for detection of the t(11;14)(q13;q32) chromosomal trans-location. DNA probe hybridization quality was compared between the tissue and isolated nuclei. Individual tumor cell nuclei were successfully extracted from each of the tissue blocks. The t(11;14) trans-location was detected by FISH in all of the samples diagnosed as mantle cell lymphoma. The hybridization signals found in the nuclei of extracted tumor cells were bright, planar, and easily identified. Detection of signal was superior to that on whole tissue samples, where signals often overlapped or were truncated. This technique produces intact nuclei for analysis, preserves the tissue block for additional studies, and allows sampling of a specific area of the tissue block. This approach may be particularly useful when the amount of diagnostic tissue is limited.

Cell Nucleus↗

PubMed1997

Novel enzyme immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue.

Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molecules could be achieved by binding the DNA onto a silica column before further purification. Viral DNA sequences could be amplified by PCR in DNA extracted from routinely processed paraffin blocks from cases with clinically or morphologically suspected cytomegalovirus or Epstein-Barr virus infections. The PCR products were specified by a novel liquid hybridization assay called PCR-enzyme-linked immunosorbent assay. Using this assay, the time-consuming Southern hybridization could be replaced and the time requirement for the detection of PCR products could be reduced from 1 day to 4 hours. The assay system described here represents a reliable, sensitive, and specific method for the detection of viral DNA from paraffin-embedded tissue samples.

Cytomegalovirus↗

PubMed2001

Immunohistological study of the immune system cells in paraffin-embedded tissues of conventional pigs.

The distribution of different cells of the immune system has been studied in formalin-fixed paraffin-embedded tissues from conventionally reared healthy pigs, using immunohistological techniques. The samples collected were: lungs, tonsils, lymph nodes (mediastinal, mesenteric, inguinal and submandibular), pancreas, spleen, liver, kidney, adrenal gland, ileum and stomach. A total of six primary antibodies anti-CD3, anti-CD79alpha, Mac 387, anti-lysozyme, anti-CD45RA (3C3/9) and anti-SLA-II-DQ (BL2H5) were used with a standard avidin-biotin peroxidase (ABC) method. Anti-CD3 and anti-CD79alpha mAb-reacted, respectively with cells located in T cell areas and B cell areas. Mac 387 recognised circulating polymorphonuclear leukocytes, while anti-lysozyme-stained resident macrophages in all tissues. 3C3/9 and BL2H5, were assessed in formalin-fixed paraffin-embedded tissues for the first time. 3C3/9 identified B lymphocytes, in primary follicles and mantle zones, a subpopulation of T cells, especially located in the marginal zone of the spleen and a variable number of immunoblasts, in the germinal centres. BL2H5 reacted with B cells in the mantle zones of the follicles of lymphoid tissues, with dendritic and interdigitating cells in all studied lymphoid tissues and with a variable number of resting and activated T cells in the periarteriolar lymphoid sheath (PALs), marginal zone and red pulp of the spleen. Furthermore, it stained Kupffer and perivascular macrophages in the liver. This study represents a detailed histological study of the distribution of the most important subpopulations of immune system cells in conventional, healthy pigs. In our view, these tools should be useful for future comparative studies in disease conditions.

Animals↗

PubMed1997

[Study of detecting p53 gene of laryngeal carcinoma on paraffin-embedded tissue sections by in situ polymerase chain reaction].

In order to investigate alterations of p53 gene in laryngeal carcinoma, five cases of paraffin-embedded tissue were detected by in situ polymerase chain reaction (PCR). The results showed that p53 gene was presented in more than 90% of cancer cells in all cases with exon 5 specific primers, which signal copy numbers were 2.24 +/- 0.23. It suggests that in situ PCR and paraffin-embedded tissue are able to be used in the single copy gene research. The improvement and application of the technique were discussed.

Carcinoma, Squamous Cell↗