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At least 55 records · Page 3Linked to original sources
PubMed1988

Dehydrogenase enzyme histochemistry on freeze-dried or fixed resin-embedded tissue.

A method has been developed for the histochemical demonstration of a variety of dehydrogenases in freeze-dried or fixed resin-embedded tissue. Seven dehydrogenases were studied. Lactate dehydrogenase, NADH dehydrogenase and NADPH tetrazolium reductase were all demonstrable in sections of paraformaldehyde-fixed resin-embedded tissue. Freeze-dried specimens were embedded, without fixation, in glycol methacrylate resin or LR Gold resin at either 4 degrees C or -20 degrees C. All the dehydrogenases except succinate dehydrogenase retained their activity in freeze-dried, resin-embedded tissue. Enzyme activity was maximally preserved by embedding the freeze-dried tissue specimens in glycol methacrylate resin at -20 degrees C. The dehydrogenases were accurately localized without any diffusion when the tissue sections were incubated in aqueous media. Addition of a colloid stabilizer to the incubating medium was not required. Freeze-drying combined with low-temperature resin embedding permits accurate enzyme localization without diffusion, maintenance of enzyme activity and excellent tissue morphology.

Animals↗
PubMed1994

Applications of DNA flow cytometry and fluorescence in situ hybridization using a chromosome-specific DNA probe on paraffin-embedded tissue sections of primary malignant melanomas.

We have applied DNA flow cytometric analysis to paraffin-embedded tissue sections of primary malignant melanomas. Conventionally, flow cytometric analysis of paraffin-embedded tissue sections has been done by the method of Hedley et al. We added ultrasound treatment to the method of Hedley et al. and a lower value of coefficient of variation was shown. Furthermore, a new technique, fluorescence in situ hybridization with a chromosome-specific repetitive DNA probe, was used for the analysis of chromosomal numerical aberrations in the same paraffin-embedded tissue sections. The DNA flow cytometric analysis showed that in 8 cases six primary malignant melanomas were of the aneuploid pattern and two cases of lentigo maligna (melanoma in situ) were of the diploid pattern. By fluorescence in situ hybridization, the two cases with the diploid pattern had spots/nucleus of 1.28 and 1.12, and those with the aneuploid pattern had spots/nucleus from 2.01 to 2.27. Only one nodular melanoma in an aneuploid case showed spots/nucleus of 1.71. These data indicate that fluorescence in situ hybridization with chromosome-specific repetitive DNA probes can serve as a cytogenetic tool for the analysis of interphase nuclei of solid human tumors and may be useful for the study of tumor cell heterogeneity.

Adult↗
PubMed1977

Electron spin resonance spectra of chicken liver and hepatoma tissue embedded in paraffin.

ESR spectra of chicken liver and hepatoma tissue embedded in paraffin display signals characteristic of free radicals and paramagnetic complexes. Owing to embedding the spectrum is altered as compared to that of the native tissue but remains characteristic of the respective tissues. The spectra of tissues embedded in paraffin allow no more than qualitative conclusions to be drawn concerning the spectra of the native tissues, even if appropriate controls are applied.

Carcinoma, Hepatocellular↗
PubMed1990

Immunohistochemical detection of c-myc oncoprotein in paraffin embedded tissues.

In almost all studies using paraffin embedded tissue, c-myc protein has been found in the cytoplasm of cells. Since the protein is normally localized in the nucleus it is difficult to determine which histochemical observations are real and which are artefactual. The study designed here evaluated several different methods of fixation prior to paraffin embedding in an attempt to identify which would prevent the diffuse of the protein out of the nucleus. Using various fixation procedures (formalin, paraformaldehyde, B-5, Zamboni and AMeX) we found that fixation in cold acetone (-20 degrees C) overnight followed by 2x15 min fixation in acetone at +4 degrees c and at room temperature, cleared in methyl benzoate and xylene (AMeX procedure) gives reproducible nuclear staining when a variety of normal and tumor tissues are treated with an anti c-myc protein antibody. This method was then compared to frozen sections. While there was no cytoplasmic staining in same tissue specimens in both AMeX processed and frozen sections, the tissue architecture was much better preserved in AMeX processed samples. Our data strongly suggest that AMeX fixation, originally developed for T and B lymphocyte antigens, should be used for immunolocalization of c-myc oncoprotein in paraffin embedded tissues.

Cell Nucleus↗
PubMed2000

Methacarn fixation: a novel tool for analysis of gene expressions in paraffin-embedded tissue specimens.

To establish a quantitative method for analysis of gene expressions in small areas of tissue after paraffin embedding, preliminary validation experiments with RT-PCR and Western blotting were performed using methacarn-fixed rodent tissues and a cultured PC12 cell line. A total RNA yield of 52 +/- 15 ng/mm2, sufficient for a quantitative RT-PCR of many genes, could be extracted from a deparaffinized 10-microm-thick rat-liver section by a simple, single-step extraction method. The low concentration of contaminating genomic DNA and the resolution of ribosomal RNAs in RNA gel proved the purity and integrity of the extracted RNA samples, allowing PCR amplification of a long mRNA sequence and mRNA species expressing low copy numbers. PCR amplification of mRNA-derived target gene fragments could be achieved by optimizing the amount of total RNA for reverse transcription and the number of subsequent PCR cycles for each gene. By this validation, organ- and sex-specific mRNA expression could be detected in methacarn-fixed paraffin-embedded tissues without additional DNase treatment of RNA samples. RT-PCR analysis could also be performed with total RNA extracted from deparaffinized tissue dissected with a laser capture microdissection system. In addition, extraction of protein yielded 4.9 +/- 2.1 microg/mm2 from a 10-microm-thick rat-liver section, allowing a quantitative expression analysis of protein by Western blotting. Thus, in addition to its advantages for immunohistochemistry, methacarn-fixed paraffin-embedded tissue has benefits for analysis of both RNAs and proteins in the cells of histologically defined areas.

Acetic Acid↗
PubMed2000

CD10 expression in follicular lymphoma versus reactive follicular hyperplasia: evaluation in paraffin-embedded tissue.

The differentiation of reactive follicular hyperplasia (RFH) from follicular lymphoma (FL) may be a diagnostic challenge and require the use of adjuvant studies. Antibodies to CD10 have been used with flow cytometry and frozen tissue sections and recently have become available for use with paraffin-embedded tissue. We sought to investigate the utility of CD10 in the differentiation of RFH from FL in paraffin-embedded tissue. Twenty-three cases of FL and 19 cases of RFH were evaluated after immunostaining with antibodies to CD3, CD20, BCL2, and CD10 antigens. Intensity of CD10 expression was graded. CD10 expression was present in 87% of cases of FL. Follicles demonstrated strong positivity in 70%. One CD10-positive case of FL showed equivocal staining for BCL2 and one an absence of staining for BCL2. Weaker but definite staining was present in 17%, and 13% were negative for CD10. In contrast, none of the cases of RFH was strongly positive for CD10 expression, with 26% demonstrating weak immunostaining, one case showing equivocal staining, and 68% being entirely negative. CD3, CD20, and BCL2 stained appropriately in all cases. These findings indicate that immunohistochemical staining of paraffin-embedded tissue sections for CD10 may help differentiate reactive from neoplastic follicular lesions.

Antigens, CD20↗
PubMed1985

Flow cytometric determination of DNA ploidy level in nuclei isolated from paraffin-embedded tissue.

Nuclei, isolated from paraffin-embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA-derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the formalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffin-embedded normal and tumor tissue of the same specimen were mixed. With this method DNA indices (DI) of 24 colorectal cancers were found to be closely correlated (r = 0.9877, P less than 0.001) with DI obtained with fresh tumor tissue from the same patients. The correlation of the percentages of S-phase nuclei between paraffin-extracted and fresh samples (r = 0.5875, P less than 0.05) was as high as could be expected, taking sampling differences into account. This method is an important tool for the retrospective analysis of FCM-derived DNA parameters in relation to diagnosis and prognosis of neoplasms.

Animals↗
PubMed1978

Immunofluorescence studies of canine distemper encephalitis on paraffin-embedded tissue.

A paraffin-embedding technique for fluorescent antibody studies of canine distemper encephalitis was developed. Specific fluorescence was demonstrated in all brain tissue from dogs with canine distemper, and when compared with tissue on cryostat sections, the paraffin-embedded tissue showed superior preservation of tissue architecture. The preservation of viral antigen was good, and the appearance of fluorescence in gray- and white-matter lesions was described. In gray matter, extensive fluorescence was found mainly in neurons; fluorescence in white matter was less extensive and was mainly associated with astrocytes.

Animals↗
PubMed1995

Isolation of nucleic acid from paraffin embedded tissue for PCR amplification and sequencing of TcR V beta genes.

PCR analysis of DNA and RNA from fresh and frozen tissue is now a routine practice in most laboratories. Furthermore DNA extracted from paraffin embedded tissue is routinely used for clonality studies and for HLA typing. However the use of RNA from paraffin embedded tissue is more problematical due to degradation and only short sequences are suitable for amplification. In this report we examine the suitability of extracted nucleic acid from paraffin embedded tissue for the PCR amplification of TcR V beta genes and sequencing of resultant PCR products.

Amino Acid Sequence↗
PubMed1986

Immunocytochemical localization of protein antigens in large sections of tissues embedded in water-soluble embedding media.

JB4 and Immunobed are water-soluble embedding media used for embedding large blocks of tissue. Immunobed was specifically designed for immunocytochemistry because ethanol extraction of an additive in the monomer of the resin is reported to render tissue sections permeable to immunoglobulins. We have modified the manufacturer's protocol to accomplish localization of two protein antigens in tissues embedded in either JB4 or Immunobed. Luteinizing hormone-beta (LH beta) was localized in sections of rat and bovine pituitary tissues and bovine placental lactogen (bPL) was localized in sections of placentomes from bovine placentas. Sections received one of the following pre-treatments: absolute EtOH; NaHCO3 buffer, pH 6-10; EtOH followed by NaHCO3 buffer; one of several enzymes; EtOH followed by enzyme; NaHCO3 buffer followed by enzyme. Anti-LH beta stained only pituitary gonadotrophs and anti-bPL stained only placental binucleate cells, as assessed by absorption controls. Pre-treatment with enzyme was required for staining of sections, but an alkaline pH change (NaHCO3) had little or no effect. Ethanol pretreatment had little or no effect alone or in conjunction with NaHCO3 or enzyme. Sections were sufficiently thin (1.5 micron) to afford resolution of structure, but suitably large (approximately 2 cm2) to minimize problems of sampling.

Animals↗
PubMed1987

Immunophenotyping of non-Hodgkin's lymphomas using a panel of antibodies on paraffin-embedded tissues.

The use of monoclonal and polyclonal antibodies for the immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue has been limited by the fact that most antigens on lymphoid cells are denatured by histologic fixation, dehydration, and embedment. In this article the authors have analyzed a small panel of antibodies which represent exceptions to this rule, in that they identify denaturation-resistant determinants on leukocyte antigens in paraffin-embedded tissue. Monoclonal antibodies L26 [corrected] and 4KB5 label preferentially B cells, monoclonal antibody UCHL1 stains predominantly T cells, and monoclonal antibody MAC 387 reacts with granulocytes and some macrophages. A polyclonal antiserum raised against purified CD3 (T3) antigen, a T-cell-specific molecule, was also employed. This antibody panel was used to immunophenotype routinely processed tissue biopsy specimens from 61 non-Hodgkin's lymphomas (all of which had been previously phenotyped in cryostat sections). The lineage of the neoplastic cells was correctly identified in 32 of 34 (94%) cases of B-cell lymphoma, in 19 of 19 (100%) cases of T-cell neoplasm, and in 2 of 4 (50%) cases of histiocytic malignancy. It is concluded that this combination of antibodies is helpful in immunophenotyping non-Hodgkin's lymphomas when only paraffin-embedded tissue sections are available, although additional reagents of higher specificity are required to improve the identification of lymphomas.

Antibodies, Monoclonal↗
PubMed1976

Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains.

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.

Acrylates↗
PubMed1992

Detection of Cryptosporidium parvum DNA in fixed, paraffin-embedded tissue by the polymerase chain reaction.

The objective of this project was to demonstrate detection of Cryptosporidium parvum DNA in fixed, paraffin-embedded tissue using the polymerase chain reaction (PCR). DNA was purified from six samples of fixed, paraffin-embedded tissue that were histologically positive for C. parvum and used in the PCR. Previously developed oligonucleotide primers specific for C. parvum were used to amplify a 452-base target sequence, and a 20-base synthetic probe labeled with digoxigenin-11-dUTP was used to detect the amplification product by chemiluminescence. All six samples were positive by PCR; negative controls showed no amplification or hybridization. This approach could provide a sensitive and specific method for detection of parasite material in fixed, paraffin-embedded tissue samples, and prove to be of significant value in retrospective studies of archival material.

Animals↗
PubMed1996

Detection of Mycobacterium tuberculosis in formaldehyde solution-fixed, paraffin-embedded tissue by polymerase chain reaction in Pott's disease.

STUDY DESIGN: Twenty-five formaldehyde solution-fixed, paraffin-embedded tissue blocks from vertebral biopsy specimen materials with presumptive diagnosis of tuberculous spondylitis and nonspecific vertebral osteomyelitis were studied. OBJECTIVES: To evaluate the sensitivity and specificity of polymerase chain reaction in detecting Mycobacterium tuberculosis in formaldehyde solution-fixed, paraffin-embedded tissue samples from histologically proved tuberculous spondylitis. SUMMARY OF BACKGROUND DATA: Diagnosis of a mycobacterial infection is a long and tedious process; because of the slow growth rate of mycobacteria on solid media, identification and antibiotic sensitivity testing can take up to 10 weeks, but the sensitivity of culture can be as low as 50%. Direct microscopy is insensitive because clinical samples may contain only few organisms. Recently, polymerase chain reaction has been applied in the rapid amplification and identification of many organisms, including mycobacteria. METHODS: The DNAs were extracted from 25 paraffin-embedded tissue blocks. An insertion element IS 6110 (Integrated DNA Tec. Inc., Corrallville, IA), a DNA sequence unique to Mycobacterium complex (M. tuberculosis and the subspecies Mycobacterium bovis), was amplified by polymerase chain reaction. Polymerase chain reaction results were compared with those of Mycobacterium culture, acid-fast bacilli staining, and histologic findings. RESULTS: Polymerase chain reaction was positive in 18 cases of 19 tuberculous spondylitis. Three of the polymerase chain reaction test results were positive with concomitant negative culture and positive acid-fast bacilli staining. There were six chronic nonspecific infections, and polymerase chain reaction results were negative in five cases; in the single positive case, DNA amplification results remained positive even after three repeated tests. CONCLUSION: Polymerase chain reaction has a sensitivity of 94.7%, specificity of 83.3%, positive predictive value of 94.7%, and a negative predictive value of 83.3%. Accuracy was calculated as 92%.

Fixatives↗
PubMed2000

Direct multiplex amplification of DNA from a formalin fixed, paraffin wax embedded tissue section.

The extraction of DNA from formalin fixed, paraffin wax embedded tissue can be problematical, with long protocols producing low yields. This report describes a very simple and useful method for amplifying DNA from formalin fixed, paraffin wax embedded tissue without the need for prior DNA extraction. This method allows direct polymerase chain reaction (PCR) based molecular analysis of fixed tissue. It is an invaluable method if clinical biopsy specimens are to be investigated, because extraction of uncontaminated DNA from such small samples can be very difficult or even impossible. It will also facilitate the study of intratumour heterogeneity, with the analysis of multiple small areas from within a single tumour section. In addition, this method can be used for other samples where only a few tests are to be carried out and a stock of DNA is not required, thus shortening the analysis time.

Biopsy↗
PubMed1994

Effects of fixative and fixation time on the extraction and polymerase chain reaction amplification of RNA from paraffin-embedded tissue. Comparison of two housekeeping gene mRNA controls.

A number of reports have indicated that RNA recovered from paraffin-embedded tissue can be used as a substrate in the polymerase chain reaction (PCR). Although it is established that RNA in paraffin-embedded tissue undergoes significant degradation, the specific contributions of different fixatives and fixation times to this degradation are not known. Mouse splenic tissue was harvested and fixed immediately for 2, 8, or 24 h in either formalin, Omnifix II, or Carnoy's fixative and then processed and embedded in paraffin. RNA was extracted from deparaffinized cubes of tissue using an adaptation of the technique described by Chomczynski and Sacchi. RNA was reverse transcribed using a random hexamer primed reaction. PCR amplification for cDNAs of the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase (HPRT) mRNAs was then performed. Although GAPDH amplification is used routinely on fresh and frozen tissues, we show that the presence of DNA contamination in the RNA preparations limits its usefulness in paraffin-embedded tissue. Amplifiable HPRT mRNA sequences were detected in nine of 12 samples fixed in Omnifix II, in four of 12 samples fixed in Carnoy's fixative, and in none of 12 formalin-fixed samples. Because of primer selection to preclude amplification of genomic HPRT, DNA contamination is not an issue when HPRT is amplified. Thus, HPRT represents the control system of choice for the evaluation of RNA in PET. The techniques described provide a rapid, uniform, and reproducible method of obtaining RNA from PET for molecular analysis, but they indicate limited utility for retrospective analysis of archival tissues.

Animals↗
PubMed1993

Diagnostic tools for differentiating between pleural mesothelioma and lung adenocarcinoma in paraffin embedded tissue. Part I: Immunohistochemical findings.

Specimens of 27 histologically definite mesotheliomas and 34 proven adenocarcinomas were examined with a panel of 14 antibodies: pan-epithelial antibody Lu-5, anti-keratin-18, anti-keratin-7, Ber-EP4, anti-Leu-M1, HEA-125, anti-carcino-embryonic antigen (CEA), anti-blood group-related antigens (anti-BGR A, B, H), B72.3, anti-placental alkaline phosphatase (PLAP), anti-vimentin and BMA-120 used to determine their value in the differentiation between pleural mesothelioma and lung adenocarcinoma. Lu-5, anti-cytokeratin-7 and -18, B 72.3 and PLAP reacted in a high percentage of cases with both mesothelioma and adenocarcinoma. Anti-CEA and anti-Leu-M1 did not react with any of the 27 mesotheliomas tested but showed a reaction in 75% (anti-CEA) and 66% (anti-Leu-M1) of the lung adenocarcinomas. Seventeen percent of the adenocarcinomas and 96% of the mesotheliomas showed a positive reaction with anti-vimentin. Ber-EP4 was demonstrated in all lung adenocarcinomas, but only in 2 mesotheliomas in a focal manner (7%). HEA-125 and anti-BGR A, B, H reacted with 83% (HEA-125) and 75% (anti-BGR A, B, H) of the lung adenocarcinomas. The statistical parameters, sensitivity and efficiency were estimated and a normogram for judging the diagnostic power of a single antibody for the differential diagnosis of mesothelioma versus adenocarcinoma was developed. According to this, Ber-EP4, HEA-125, anti-BGR A, B, H and anti-CEA were, in descending order, the most powerful discriminatory antibodies.

Adenocarcinoma↗