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Epithelial markers for paraffin-embedded human tissues. Immunohistochemistry with monoclonal antibodies against milk fat globule antigens.

About 200 human tumours and corresponding normal tissue samples were investigated by immunoperoxidase tests for the expression of MAM-3, MAM-5 and MAM-6 antigens, which had previously been defined by monoclonal antibodies to human milk fat globule membranes. All tissue specimens had been treated for routine histopathology, i.e. fixed in formalin and embedded in paraffin. One of the antigens, MAM-6, appeared to be an important epithelial marker, present in all normal and neoplastic breast tissue samples, in about 80% of non-mammary normal tissues and in more than 90% of non-mammary epithelial tumours. It could never be detected in normal and neoplastic mesenchymal and neuroectodermal structures. Direct comparison with the distribution of Carcinoembryonic Antigen (CEA), Tissue Polypeptide Antigen (TPA) and keratin provided clear evidence that MAM-6 is different from these well known epithelial markers. MAM-3 proved to be an additional important marker exhibiting a characteristic distribution pattern in those epithelial tissues investigated. In contrast to MAM-6, it could never be detected in renal cell cancers and carcinomas of the prostate gland, thus allowing differential diagnosis on the basis of immunohistochemistry. MAM-5, known to be associated with lactoferrin, was mainly detectable in secretory organs and their tumours. In the group of breast tumours, its expression was mainly seen in lobular cancers. These findings suggest a use for these new markers for routine histopathology.

Adenocarcinoma↗

Aldehyde Fuchsin staining of pancreatic B cells. Reproducible high-contrast staining of formalin-fixed and paraffin-embedded material.

The use of Aldehyde Fuchsin for the demonstration of B cell granules in the pancreatic islets on material fixed in formalin and embedded in paraffin has led to variable results. Treatment of such sections for 1 h with Bouin's fluid or 5% glutaraldehyde prior to deparaffinization, however, stabilizes the secretory granules in B cells. In addition, the zymogenic granules of the acinar cells exhibit increased stainability with the permanganate Aldehyde Fuchsin procedure.

Animals↗

Molecular genetic analysis of DNA obtained from fixed, air dried or paraffin embedded sources.

In the present study methods are described for the analysis of the genomic structure of DNA isolated from tissues that have been stored up to several years as air-dried or stained bone marrow smears, bone marrow biopsies in "Histicon", chromosomal preparations in fixative or as formalin fixed tissues embedded in paraffin. By application of the new techniques clonal B- and T-cell disorders, Philadelphia chromosome translocations and ras oncogene mutations in pancreatic carcinomas could be detected. Thus, these DNA extraction procedures may open new avenues to pathological archives and enable the analysis of samples when fresh material is not available.

Bone Marrow↗

Purification of DNA from formaldehyde fixed and paraffin embedded human tissue.

The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.

Carcinoma↗

Immunofluorescent detection of hepatitis B antigen in paraffin-embedded liver tissue.

Hepatitis B antigen (HBAg) has been demonstrated by the indirect immunofluorescent technique and by orcein staining in 20 liver biopsies fixed in Bouin's fixative and embedded in paraffin. The results were compared with those obtained previously by immunogluorescence on frozen sections of the same biopsies. Ten biopsies which were positive in frozen sections were also positive by immunofluorescence in parafin sections, whereas only six were positive by orcein staining. In orcein-stained sections, the cellular localization of HBAg was precisely in the same places as in the slides examined by immunogluorescence. The intessity of the fluorescence in paraffin sections was almost the same as in frozen sections. The localization of the antigen was histologically more precise in paraffin sections. Besides various advantages, indlucing aboidance of freezing aquipment and procedures, paraffin sections are more easy to handle and biopsies from distant hospitals can be processed. The advantages of the immunofluorescent test in comparison to orcein staining are its immunological specificity and higher sensitivity.

Biopsy↗

Use of the avidin-biotin peroxidase system to detect rabies antigen in formalin-fixed paraffin-embedded tissues.

We stained rabies-infected nervous and salivary-gland tissues fixed in formalin or acetone and embedded in paraffin with the avidin-biotin peroxidase system. With this system, rabies-virus antigen was detected in neurons, glandular acinar cells, and vascular endothelial cells more effectively than by immunofluorescence, especially when tissues were enzyme-digested with pronase before immunoperoxidase staining. The avidin-biotin peroxidase system should be useful for routine diagnosis, retrospective studies of rabies, and identification of specific cells involved in the spread of virus in rabies-infected hosts.

Animals↗

Direct immunofluorescence of skin using formalin-fixed paraffin-embedded sections.

The technique of direct immunofluorescence has been applied to skin biopsy specimens fixed in formalin and embedded in paraffin wax. The results have been compared with those obtained by using snap-frozen biopsy specimens from the same patients. Trypsinisation of the dewaxed material allowed subsequent detection of immunoglobulins, complement, and fibrinogen. When compared to the fluorescence in the snap-frozen specimens, the staining in the paraffin sections was less bright and there was a higher rate of negatives. Even so, it was possible to establish the diagnosis in most cases of pemphigus, pemphigoid, and lupus erythematosus.

Complement C3↗

Immunostaining of growth hormone and prolactin in paraffin-embedded and stored or previously stained materials.

In attempts to evaluate immunocytochemically autopsy and biopsy material previously obtained and processed for conventional histologic staining, we had to resort to immunostaining of tissues embedded years ago or even sections already stained with hematoxylin-eosin or aldehyde thionin-PAS-orange G. Hypophysial growth hormone and prolactin proved remarkably resistant to such prior treatment with regard to their antigenic properties, and could be readily immunostained in tissue embedded in paraffin 3-4 years earlier, and after destaining of sections prepared up to 7 years earlier. The results of such "retrospective" immunocytochemical evaluation of autopsy and biopsy materail is illustrated with the staining of "pregnancy cells" for prolactin in the hypophysis of a woman postpartum, the immunostaining for prolactin in the cells of adenomas associated with marked hyperprolactinemia, the staining for growth hormone in adenomas removed from children with gigantism, and the immunostaining for prolactin, growth hormone or both in several adenomas that were discovered at autopsy and not associated with a known clinical history of endocrine aberrations.

Adenoma, Chromophobe↗

Detection of HBs antigen in routine paraffin embedded liver tissue by enzyme-labelled antibody technique.

HB surface antigen (HBs Ag) was detected using the enzyme-labelled antibody technique on routinely processed liver biopsy material fixed in Bouin's fixative and embedded in paraffin. Of 85 examined specimens, 45 cases were HBs Ag positive by both the immunofluorescent test and the enzyme labelled antibody technique. The remaining 40 cases were negative by both techniques. The specificity of HBs Ag detected by the enzyme-labelled antibody technique was confirmed by the blocking test using guinea pig specific HBs antibody. The results indicate that the enzyme-labelled antibody technique may be useful for detecting HBs Ag on routine paraffin sections.

Humans↗

Detection of African swine fever viral antigens in paraffin-embedded tissues by use of immunohistologic methods and polyclonal antibodies.

Tissues obtained from pigs inoculated with African swine fever virus (ASFV), fixed by vascular perfusion using glutaraldehyde, and embedded in paraffin or araldite were used for an immunohistologic electron microscopic study. To detect ASFV antigens, 4 methods were used on paraffin sections with or without pretreatment of the tissues. Use of biotinylated anti-ASFV antiserum combined with avidin-biotin complex and peroxidase proved to be the most suitable method, and antigen was detected in tissues infected with 2 ASF viruses of different virulence. Use of the glutaraldehyde fixation method should ensure optimal morphologic (structural and ultrastructural) data while allowing an immunohistologic study, and add to knowledge of the pathogenesis of ASF.

African Swine Fever↗

Comparison of DNA ploidy in routine fine needle aspiration biopsy samples and paraffin-embedded tissue samples.

The nuclear DNA content was determined by flow cytometry (FCM) from unfixed fine needle aspiration (FNA) biopsy samples of 31 human tumors, and from the same tumors after their excision, fixation with formalin and embedding in paraffin. The ploidy of the histograms was the same in 29 (94%) of the 31 cases. The disagreement in two cases may be explained by clonal heterogeneity of the tumors. The DNA index of the aneuploid cases was identical in fresh and fixed samples. The coefficient of variation of the diploid peaks (P less than .001) and the mean percentage of S-phase cells (P = .06) were larger in the fixed samples. It is concluded that routine FNA biopsy is a practical and reliable method for collecting cells for FCM DNA ploidy determination.

Adenoma↗

Paraffin embedded "Echinococcus granulosus" protoscoleces as suitable antigen in the indirect immunofluorescence test for human hydatid disease.

The employment of Echinococcus granulosus protoscoleces embedded in paraffin as antigen for the indirect immunofluorescence (IIF) test for human hydatid disease is described. An evaluation of its sensitivity and specificity is made studying 108 pre-operative sera from surgically confirmed hydatidosis and 91 control sera. Using the positivity criterion based on the serological reactivity at which minimum cross-reactivity was observed, the IIF test showed an overall sensitivity of 83.3% and 2.2% of cross-reactions. No differences in sensitivity between sera from hepatic and pulmonary hydatidosis was observed. The advantages of the method are pointed out.

Cross Reactions↗

[HOX11L2 expression and its clinical significance in paraffin-embedded T-lymphoblastic lymphoma].

OBJECTIVE: To determine the feasibility of detecting target gene expression and evaluate the expression of homeobox gene HOX11L2 as well as its clinical significance in paraffin-embedded T-lymphoblastic lymphoma T-LBL. METHODS: HOX11L2 mRNA was analyzed by RT-PCR and real-time fluorescent quantitative PCR (RQ-PCR) on paraffin-embedded tissues in 54 cases of T-LBL. RESULTS: HOX11L2 expression was observed in 7 cases (13.0%). The age ranged from 5 to 15 years, with a median of 9 years. All of the HOX11L2 expressed patients had prior mediastinal masses, and 5 of them expressed high level of Ki67 antigen (PI> or =80%). Symptoms of CNS invasion were diagnosed in 2 cases. Significant differences in the age ratio (P < 0.05), the level of Ki67 antigen (P < 0.05) and the incidence of mediastinal involvement (P < 0.05) were observed between patients positive or negative for HOX11L2. Patients positive for HOX11L2 had a worse outcome than those negative for HOX11L2, with median survival of 7 and 11.5 month, respectively. No statistic difference was found in sex, clinical stages, CNS and bone marrow involvement between HOX11L2 positive and negative groups. CONCLUSIONS: Detection of target gene expression in paraffin-embedded tissues is feasible by RT-PCR or RQ-PCR. HOX11L2 expression was limited in childhood with T-LBL and probably associated with the development and poor prognosis of some pediatric T-LBL patients.

Feasibility Studies↗

[The detection of viral nucleic acid sequences in paraffin-embedded subacute sclerosing panencephalitis (SSPE) and progressive multifocal leukoencephalopathy (PML) brain tissues by in situ hybridization].

We have been able to detect the viral nucleic acid sequences in formalin-fixed paraffin embedded tissue sections of the brain from SSPE and PML respectively by in situ hybridization using cloned and radiolabelled complementary DNA (cDNA) measles nucleocapsid (NP) and JC virus probes which are gene specific respectively. We have mainly followed in situ hybridization procedure reported by Haase et al, but partly modified the technique including the extensive wash by buffer solution and the elevated concentration of proteinase K. Firstly, to compare with the number of hybridized cells and grain counts, the in situ hybridization were performed in SV 40 infected cells treated with ethanol-acetic acid for 15 minutes or 10% neutral formalin for at least one week to simulate procedure used by pathologists. Similar findings were obtained in both treatment of 1 microgram/ml concentration of proteinase K in ordinary fixed cells and in 100 micrograms/ml in formalin-fixed cells. In addition we investigated the ability of in situ hybridization technique to detect measles in SSPE and papova virus in PML obtained postmortem that were been formalin-fixed paraffin-embedded routinely. In paraffin-embedded SSPE cerebral cortex positive hybridization were obtained in oligodendroglias and neurons using 125I-labelled measles NP probe in 6 days' autoradiographic exposure (Fig. 2 and 3) in our modified technique. Additionally, in PML tissue JC virus nucleic acid sequences were also detected in many oligodendroglias and swollen neurons using 3H-labelled JC virus probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

DNA flow cytometric values in bladder carcinoma biopsies obtained from fresh and paraffin-embedded material.

DNA-histograms were obtained by flow cytometry (FCM) of nuclei prepared from fresh bladder carcinoma biopsies and from 100 micron sections of paraffin-embedded material from the same biopsies. Linear regression analysis of ploidy values from fresh material versus those from paraffin blocks showed excellent correlation (R2 = 0.957). Owing to the high background, the paraffin-embedded material was less suited for analysis of S-phase fraction. It is concluded that DNA FCM of nuclei obtained from paraffin-embedded bladder carcinoma biopsies yields reliable results concerning ploidy, but does not permit evaluation of the S-phase fraction.

Biopsy↗

Detection of p53 overexpression in routinely paraffin-embedded tissue of human carcinomas using a novel target unmasking fluid.

With the aid of a newly developed target unmasking fluid (TUF), p53 overexpression was visualized by immunohistochemistry on recent and archival paraffin-embedded tissue samples of colon, stomach, and pancreas neoplasms. Using monoclonal anti-p53 antibody pAb1801 as well as polyclonal antiserum to p53 CM1, TUF-mediated immunohistochemistry was fully concordant for p53 overexpression in paraffin-embedded carcinoma samples compared with freshly frozen tissue from the same tumors. Thus, prognostic and diagnostic assessment of p53 overexpression in malignant tissue, routinely fixed in formalin and embedded in paraffin wax, by TUF-mediated immunohistochemistry with monoclonal and polyclonal antibodies may be adopted as a new tool in diagnostic and research histopathology.

Antibodies↗

[Flow cytometric analysis of DNA content in paraffin-embedded tissue in head and neck cancer (the second report)--primary lesions of laryngeal cancer].

Flow cytometric analysis of DNA content using paraffin-embedded materials has become an important diagnostic, as well as prognostic, method for clinical pathology and investigative oncology. DNA content as measured in paraffin-embedded materials is closely related with that obtained from fresh specimens. This method also permits retrospective analysis on a lot of cases and studying different specimens of a tumor for intratumor heterogeneity. Flow cytometry was used to investigation of the DNA distribution in biopsy specimens from 30 patients with squamous cell carcinoma of the larynx. These patients had initially treated in our institute in the period from 1986 to 1988 and followed for two to four years. A modification of Hedley's method was used to prepare paraffin-embedded materials. DNA histogram were assessed in terms of DNA index (DI). The coefficient of variation for determination of DI ranged 4.2 to 13.7% (mean 7.8 +/- 2.4%). Using infiltrated lymphocytes and mesenchymal cells as an internal standards, aneuploid DNA histogram were found in 13 of 30 specimens (43.3%). DI ranged from 1.05 to 1.22. There was no significant correlation between DNA content and sex, age, tumor size, staging or treatment. The aneuploidy was found in 2 of 17 cases with well differentiated tumor (11.8%), 10 of 12 with moderately differentiated tumor (83.3%), one with poorly differentiated tumor (100%). 11.8% of patients with diploid tumor cells had recurrence during the interval of observation, compared to 38.5% of those with aneuploid tumor cells (p less than 0.05). In T1 lesions of glottic cancer aneuploid group had a significantly lower local control rate than diploid group (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

A microwave method that enhances detection of aberrant p53 expression in formalin-fixed, paraffin-embedded tissues.

OBJECTIVE: Detection of aberrantly accumulated p53 protein by immunohistochemistry may have prognostic significance in many human neoplasms. We wished to identify a technique applicable to formalin-fixed, paraffin-embedded sections that would reliably yield results equivalent to frozen-section immunohistochemistry. DESIGN: We compared the frequency of p53 immunostaining obtained by applying monoclonal antibodies PAb1801, DO7, or DO1, a 1:1 PAb1801-DO7 cocktail, and a 1:1 PAb1801-DO1 cocktail to fresh-frozen and formalin-fixed, paraffin-embedded tissues from 36 lung and upper aerodigestive-tract carcinomas. With the formalin-fixed tissues, we compared pepsin predigestion with microwave irradiation in citrate buffer as means of enhancing the sensitivity of p53 detection. SETTING AND PATIENTS: All tissues were obtained from surgical resections of tumors, from patients who underwent surgery at the Minneapolis Department of Veterans Affairs Medical Center between 1990 and 1992. MAIN OUTCOME MEASURES: The sensitivity of different paraffin section techniques for immunohistochemical detection of accumulated p53 protein was determined in reference to the optimal frozen section method (defined as the method that yielded the greatest number of p53-positive cases in frozen tissue). RESULTS: Microwave antigen retrieval markedly enhanced staining with PAb1801 and DO7 in paraffin sections, as compared with pepsin predigestion and no pretreatment. This technique was 100% sensitive relative to the optimal frozen tissue method. In contrast, staining with DO1 alone was not enhanced by microwaving. CONCLUSIONS: Microwave pretreatment in conjunction with the use of either PAb1801 or DO7 is highly efficacious in the immunohistochemical detection of aberrant p53 expression in formalin-fixed, paraffin-embedded tissues.

Antibodies, Monoclonal↗