SEARCH · PubMed Health

Results for “Paraffin Embedding”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 289 records · Page 16Linked to original sources

PubMed1986

DNA flow cytometry of cells obtained from old paraffin-embedded specimens. A comparison with results of scanning absorption cytometry (a methodological study).

Suspensions of single cell nuclei were obtained from 31 different samples of 7-9 years old paraffin-embedded bladder cancer biopsies. The DNA content of the ethidium-bromide-stained nuclei was analyzed by flow cytometry (FCM). The tumour stemline ploidy, as determined by FCM, was compared with that obtained by Feulgen scanning absorption cytometry (SACM) in imprints obtained from the same biopsy before it was fixed. Fifteen tumours that were diploid by FCM were also diploid by SACM. All of the 16 tumours with non-diploid DNA-stemlines by FCM were non-diploid by SACM, though minor differences between the ploidy values were occasionally seen when the results of the two methods were compared. The background activity due to cell debris was considerable and resulted in a mean variation coefficient (CV) of 5.1% for human spleen cells fixed and embedded before preparation for FCM in the same way as the tumour samples. In the tumour samples there was a large and unpredictable variation of the ratio between the DNA values of chicken erythrocytes (internal standard) and diploid cells. In some specimens this ratio would have resulted in incorrect evaluation of the FCM DNA histogram. The following method for evaluation of FCM histograms is therefore proposed: Single peak histograms obtained from paraffin embedded tissue should always be interpreted as representative for a diploid cell population. In FCM histograms from paraffin-embedded tissue with more than one peak, the first peak should be considered as representing diploid cells.

Animals↗

PubMed1996

Retrieval of leukocyte antigens in paraffin-embedded rat tissues.

We compared the immunohistochemical reactivity of various rat leukocyte antigens in frozen and paraffin-embedded thymus, spleen, abdominal lymph node, liver, and brain tissues of healthy Sprague-Dawley rats, fixed in various fixatives. Immune reactivity after fixation in Methacarn was superior to that of 4% neutral buffered formalin, a mercury-based fixative (B-5), or Carnoy. Microwave (MW) antigen retrieval (AR) enhanced antigen reactivity. Ten of the 11 leukocyte antigens studied could be retrieved in Methacarn-fixed, paraffin-embedded sections with a reactivity comparable to that obtained on frozen sections.

Animals↗

PubMed1976

A new metachromatic stain technique for paraffin-embedded neural tissue using thionin.

A metachromatic staining procedure which differentiates Nissl substance in neuron cell bodies, neuropil area, and axonic fibres of passage with distinct colours with no counterstaining has been developed for paraffin-embedded tissue utilizing thionin stain. Frozen sections of neural tissue fixed in formalin may also be satisfactorily stained by this procedure, and both paraffin-embedded and frozen sections have been found to retain the distinct colours for a year with little fading. Since metachromatic staining of paraffin-embedded neural tissue has not been successfully achieved before, the following procedure will be especially valuable for studying small vertebrate brains, or other central nervous tissue which must be processed by the paraffin method.

Animals↗

PubMed1998

Comparison of three different primer pairs for the detection of Mycobacterium tuberculosis by polymerase chain reaction in paraffin-embedded tissues.

SETTING: More than five different primer pairs have been used for the detection of Mycobacterium tuberculosis deoxyribonucleic acid (DNA) with the polymerase chain reaction (PCR). OBJECTIVE: The sensitivity and specificity of PCR were evaluated using three different primer pairs in the detection of M. tuberculosis in paraffin-embedded tissues. DESIGN: Thirty-eight tissue specimens from 23 patients were studied. Eighteen samples were obtained from 10 tuberculosis patients, and 20 samples obtained from 13 patients with other diseases were used as negative controls. DNA extracted from paraffin-embedded tissues was used directly for PCR amplification using primers IS1 and IS2 to amplify a 123 base pair (bp) region of IS6110, sjMT3 and sjMTr2 to amplify a 281 bp region of protein antigen b, and INS1 and INS2 to amplify a 245 bp region of IS986. Each amplification was performed double-blinded and repeated three times including positive and negative control samples. RESULTS: IS1 and IS2 gave a positive result in each of the double samples obtained from eight tuberculosis patients and in the single samples obtained in the two others, sjMT3 and sjMTr2 detected 13 of the 18 tuberculosis samples, and INS1 and INS2 detected only three of the 18. CONCLUSION: These results highlight the importance of selecting appropriate primers to obtain high sensitivity in detecting M. tuberculosis in paraffin-embedded tissues by PCR.

Case-Control Studies↗

PubMed2006

Multiparameter immunofluorescence on paraffin-embedded tissue sections.

Immunohistochemical techniques have gained increasing importance in diagnostics and research. While formalin-fixed, paraffin-embedded human tissue retains excellent morphology, the detection of antigens by immunofluorescence in its sections and especially the demonstration of multiple simultaneous antibodies have limitations. Double immunofluorescence labeling of routinely processed paraffin sections has been described previously. The signal intensity observed after triple labeling has been reported to be significantly inferior to that obtained by application of double fluorochromes. The authors show multicolor labeling of three and four primary antibodies in routinely processed paraffin-embedded tissue sections using a standardized immunofluorescence technique. In addition, procedures to reduce background staining and to avoid nonspecific double staining are described.

Antibodies↗

PubMed2005

Double labeling and simultaneous detection of B- and T cells using fluorescent nano-crystal (q-dots) in paraffin-embedded tissues.

A double immunohistochemical technique for the simultaneous detection of T- and B cells in paraffin-embedded mice tissues have been developed. This procedure is based on using fluorescent nano-crystals (q-dots). The benefit of using q-dots evolves from their unique fluorescence characteristics advantages: such as broad excitation spectrum, narrow emission band and high photo-bleaching threshold compare to organic fluorophores. T cells antigens (CD3) were stained using antibody-coated q-dots with max emission at 655 nm (GalphaRb-QD655). B cells antigens (CD45R/B220) were stained using streptavidin-coated q-dots with max emission at 585 nm (SA-QD585). The simultaneous detection of T- and B cells was demonstrated in paraffin-embedded lymph node using standard fluorescence microscope.

Animals↗

PubMed2000

T311--an anti-tyrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues.

Tyrosinase is a key enzyme in melanin biosynthesis and represents a marker of melanocytic differentiation. We previously generated T311, a murine monoclonal antibody to the tyrosinase recombinant protein. This study was performed to evaluate T311 as a diagnostic immunohistochemical reagent for use on formalin-fixed paraffin-embedded pathological material. We analyzed the specificity of the antibody on a panel of normal and neoplastic tissues, and we assessed its sensitivity in a large number of metastatic and primary malignant melanomas, nevi, three angiomyolipomas, and two vitiligo specimens. T311 revealed intense reactivity on paraffin-embedded material. Immunoreactivity was limited to cells of melanocytic differentiation and no immunostaining was present in unrelated normal tissues and tumors. Eighty-four percent of metastatic malignant melanomas were immunoreactive with T311 and showed predominantly a homogeneous expression pattern. However, in primary melanomas of the desmoplastic/spindle cell type, T311 revealed a poor immunoreactivity. Nevi showed intense staining at the junctional zone, while the dermal component revealed decreasing reactivity towards deeper areas. Only one angiomyolipoma was focally immunoreactive with T311. Vitiligo specimens were immunonegative. We conclude that T311 is a specific and sensitive marker for the detection of melanocytic lesions in formalin-fixed paraffin-embedded tissues and a useful serological reagent for diagnostic pathology.

Antibodies, Monoclonal↗

PubMed1997

The in situ hybridization and immunocytochemistry techniques for characterization of cells expressing specific mRNAs in paraffin-embedded brains.

In situ hybridization (ISH) represents a powerful and sensitive method for examining gene expression in individual cells in a manner analogous to immunocytochemistry (ICC) which is used to localize and identify cells containing a particular protein or labile antigens such as amino acid neurotransmitters. Considerable progress has been made both in molecular cloning which provides nucleotide probes and in the production of antibodies to specific antigens, especially those directed against small haptenes such as GABA and catecholamines. Consequently, specific tools have become available for ISH and ICC procedures in most cases. Several techniques including double ISH, double ICC and ISH coupled to ICC have been developed to characterize the phenotype of cells expressing neurotransmitters or specific neuroreceptors. This has been particularly well illustrated for neuropeptides (Young, III, W.S., Mezey, E. and Siegel, R.E. Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization and histochemistry, Mol. Brain Res., 1 (1986) 231-241[17]) or dopamine receptors (Le Moine, C., Normand, E., Guitteny, A.F., Fouque, B., Teoule, R. and Bloch, B., Dopamine receptor gene expression by enkephalin neurons in rat forebrain, Proc. Natl. Acad. Sci. (USA), 87 (1990) 20-24 [8]; Le Moine, C., Normand, E. and Bloch, B., Phenotypical characterisation of the rat striatal neurons expressing the D1 dopamine receptor gene, Proc. Natl. Acad. Sci. (USA), 88 (1991) 4205-4209 [9]) but exclusively on frozen tissues. Combination of ISH and ICC allows the simultaneous detection of specific mRNAs and antigens in a same section or in adjacent sections. However, when ISH is coupled to ICC on a same section, a loss of signal in ISH is often observed (Siegel, R.E., Localization of neuronal mRNAs by hydridization histochemistry. In: P.M. Conn (Ed.), Methods in Neurosciences. Gene Probes. Vol. 1, Academic Press, New York, 1989, pp. 136-150 [13]) which may be a handicap when the rate of mRNA expression is low. Moreover, interferences between ISH coupled to ICC techniques may occur leading to artifactual signals. In the present protocol, in situ hybridization with a specific 35S-labeled riboprobe and immunocytochemistry with a polyclonal antibody are performed on adjacent sections of paraffin-embedded rodent brains. Paraffin embedding allows a better preservation of tissue morphology than freezing procedure. This is of particular importance since simultaneous detection of mRNAs and proteins in the same cell populations on adjacent sections requires conditions where (a) cell morphology is highly retained in a same manner on adjacent sections, and (b) reactivity of nucleic acids and antigens is preserved. We have used this technique to analyze the presence of serotonin 5-HT(1B) mRNA in GABAergic cell bodies (Cloëz-Tayarani, I., Wusher, N., Huerre, M. and Fillion, G., Cellular localization of 5-HT(1B) receptor mRNA in the rat olfactory tubercle: do GABAergic neurons express the 5-HT(1B) gene? Mol. Brain Res., 36 (1996) 337-342 [3]).

Animals↗

PubMed1993

Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections.

Enzymatic and non-enzymatic treatments for antigen unmasking on formalin-fixed, paraffin-embedded, dewaxed sections were optimized and compared by the use of a panel of antibodies of diagnostic relevance (anti-cytokeratins, vimentin, S-100, T- and B-cell receptors, Ki-67/MIB 1, muscle actin). Non-enzymatic unmasking was obtained by boiling the slides in a microwave oven in 0.01 M salt solution (pH 6) or in 6 M urea. Trypsin or pronase digestion was used for comparison and found to be necessary for some of the reagents. The investigation was then extended to 256 antibodies; the epitopic amino acid sequence was known for 48 of them. We found that enzymatic and non-enzymatic antigen unmasking are not dependent on the epitope sequence, but some antigens benefit selectively from one treatment but not from the other. Denaturation of proteins is the likely mechanism which leads to immunodetection on microwave oven-boiled slides; this suggestion is supported by the use of denaturating solutions and by the observation that endogenous enzymes were inactivated and a few antigens were no longer immunodetectable after boiling. Non-enzymatic methods for antigen unmasking are a powerful new tool for broadening the use of antibodies for immunostaining formalin-fixed, paraffin-embedded sections and should be used in parallel with the traditional enzymatic methods.

Antibodies↗

PubMed1994

Comparison of polymerase chain reaction and immunohistochemistry for the detection of Mycoplasma pulmonis in paraffin-embedded tissue.

The polymerase chain reaction (PCR) is a reliable method of detecting specific DNA sequences. The purpose of this study was to develop a PCR method of detecting Mycoplasma pulmonis in paraffin-embedded tissue sections and to compare the sensitivity of this method with that of the avidin-biotin complex (ABC) immunohistochemistry technique. We infected mice intranasally with 10(7) M. pulmonis and sacrificed them 28 days later. Using a published oligonucleotide primer sequence for M. pulmonis, we examined 8-microns paraffin sections by PCR for a 710 base-pair segment of the genome. A sequential 8-microns paraffin section was stained for M. pulmonis, using standard ABC immunohistochemistry methods. By PCR and dot blot hybridization, 60 of 62 paraffin-embedded lungs were positive for M. pulmonis, whereas only 17 of 62 lungs were positive, using the ABC method. In this study, we demonstrated that PCR of paraffin-embedded tissues followed by dot blot hybridization of the PCR products was much more sensitive than the ABC method in identifying mouse lungs infected with M. pulmonis.

Animals↗

PubMed2003

A microdissection and molecular genotyping assay to confirm the identity of tissue floaters in paraffin-embedded tissue blocks.

CONTEXT: A recurring problem in surgical pathology practice is specimen mix-up and floater contamination. While many cases can be resolved histologically, a significant number remain unclear and may have serious clinical and medicolegal implications. OBJECTIVES: To design a microdissection and genotyping assay to identify contaminating floater tissues in paraffin-embedded tissues that is optimized for small samples, and to use the assay to resolve a series of clinical cases with floater tissues. MATERIALS AND METHODS: Twenty-one cases of possible tissue floater contamination in paraffin-embedded tissue blocks were included. Using 4 unstained, 4-microm-thick histologic sections, multiple sites were microdissected under direct visualization either by hand or by laser capture microdissection. Nonneoplastic and neoplastic tissues were sampled. Polymerase chain reaction was performed for a panel of 10 polymorphic microsatellite markers at 1p34, 3p26, 5q21, 9p21, 10q23, and 17p13. Allele size and content were analyzed semiquantitatively by fluorescent capillary electrophoresis, and the genotypes for the tissues in the paraffin-embedded tissue blocks were compared for identity. RESULTS: Tissue identification was successful in all cases, despite small tissue sample size and fixation effects. Comparative analysis of neoplastic tissue floaters and the presumptive source tumor was performed when possible to control for possible allelic loss or microsatellite instability. CONCLUSIONS: Microdissection and genotyping are effective and reliable means to objectively resolve problems of possible floater contamination. Even minute tissue samples provide sufficient DNA template for polymerase chain reaction microsatellite analysis. Because of the potential clinical implications of floaters, we recommend that all suspected floaters that would change a diagnosis from benign to malignant be subjected to genotyping assay to confirm the identity of the floater tissue.

Biopsy↗

PubMed2001

Immunohistochemical detection of cell growth fraction in formalin-fixed and paraffin-embedded murine tissue.

Monoclonal antibody MIB-1 is a reliable tool for determining proliferating cells in human tissues, but does not react with the homologous mouse antigen and is therefore useless in experimental pathology using mice as model systems. Standard method for assessment of cellular proliferation in formalin-fixed, paraffin-embedded murine tissues is immunohistochemical detection of DNA synthesis using antibodies against exogenously injected 5-bromodeoxyuridine (BrdU), which is a tedious procedure and not useful for routine investigations. We tested monoclonal antibody MIB-5 and monoclonal and polyclonal anti-MCM3 antibodies as immunohistochemical proliferation markers for paraffin-embedded nonneoplastic and neoplastic tissues of wild-type and transgenic mice, compared to anti-BrdU immunostaining. Percentage of proliferating cells was determined with continuously decreasing antibody dilutions. Percentages of MIB-5 and anti-BrdU immunostained cells correlated strongly, as well as percentage of MIB-5-decorated cells and frequency of mitotic figures. Anti-MCM3 antibodies labeled significantly higher percentages of cells than anti-BrdU or MIB-5, and showed a linear decrease with increasing antibody dilutions. We conclude that MIB-5 detects reliably the cell growth fraction in formalin fixed, paraffin-embedded murine tissues, bypassing methodological drawbacks of BrdU. Anti-MCM3 antibodies are less useful for determination of proliferating cells although they might detect the fraction of cells remaining competent for proliferation.

Adenoma↗

PubMed1989

HBV-DNA by in situ hybridization. A method to improve sensitivity on formalin-fixed, paraffin-embedded liver biopsies.

In situ hybridization of hepatitis B virus DNA by means of biotinylated probes is a rapid specific histochemical method but it is not very sensitive in formalin-fixed, paraffin-embedded material. The specificity and sensitivity of a new method using a digitonin solution and hydrolytic bath before cytohybridization in 19 formalin-fixed, paraffin-embedded liver biopsies was evaluated and a noticeable increase in staining intensivity observed. Digitonin and hydrochloric acid at low concentration increase the permeability and the diffusion of the bio-probe, improving significantly the sensitivity of reaction. Hepatocytes containing few copies of HBV-DNA were revealed by this method and microscopic visualization of specimens with numerous viral episomes was considerably improved. Therefore cytohybridization can be a specific and sensitive method, even for formalin-fixed, paraffin-embedded material, revealing active HBV replication even in HBcAg negative hepatocytes.

DNA, Viral↗

PubMed1995

Comparison of flow and image cytometry for DNA content analysis of fresh and formalin-fixed, paraffin-embedded tissue in breast carcinoma.

DNA ploidy and S-phase fraction are considered to be prognostic variables in breast carcinoma. DNA content of 35 cases of breast carcinoma of varying histologic types and nuclear grades was analyzed by flow cytometry and image analysis in both fresh and formalin-fixed, paraffin-embedded tissue. Fresh cell and deparaffinized nuclear suspensions were used for flow cytometry. Fresh and deparaffinized tumor tissue samples were used for image analysis. The results of analysis for DNA ploidy, DNA index of DNA aneuploid Go/G1 peaks, and S-phase fraction were compared in different tissue preparations for both techniques. The two techniques produced comparable DNA ploidy results with both fresh and formalin-fixed, paraffin-embedded tissue. Sensitivity for detection of DNA aneuploidy was somewhat greater by image analysis, particularly in deparaffinized tissue. There was 89% agreement in detection of DNA aneuploidy by flow cytometry in fresh and paraffin-embedded, formalin-fixed tissue; the coefficients of variation of the DNA diploid Go/G1 peaks were much wider in the latter. In image analysis there was 91% agreement between fresh and fixed specimens. Agreement between the flow cytometry and image analysis in fresh specimens was 91%; in deparaffinized nuclear suspensions it was 94%. There is a high degree of correlation between the values of DNA index of DNA aneuploid Go/G1 peaks; the estimates of S-phase fraction are much more variable. Results also show a good correlation of the DNA ploidy with the nuclear grades.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma, Mucinous↗

PubMed2005

Gene mutation analysis in five cases of dermatofibrosarcoma protuberans using formalin-fixed, paraffin-embedded tissues.

Fusion of the collagen type I a 1 (COL1A1) gene with the platelet-derived growth factor B-chain (PDGFB) gene has been pointed out in dermatofibrosarcoma protuberans. Various exons of the COL1A1 gene have been shown to be involved in the fusion with exon 2 of the PDGFB gene. We studied the breakpoints of the COL1A1 gene using formalin-fixed, paraffin-embedded tumour specimens from five patients with dermatofibrosarcoma protuberans (three reconfirmations and two new cases). Reverse transcriptase-PCR was performed using paraffin-embedded tissues. Nucleotide sequence analysis was carried out using the PCR products to identify the breakpoints. The COL1A1-PDGFB fusion transcripts were detected from the tumour specimens. Sequence analysis revealed that the ends of exons 18, 29, 38, 42 and 44 in the COL1A1 gene were fused with the start of exon 2 in the PDGFB. This study identified a novel COL1A1 breakpoint, namely, exon 44 of the COL1A1 gene. Detection of the aberrant fusion transcript using formalin-fixed, paraffin-embedded tumour specimens is useful as a diagnostic aid for dermatofibrosarcoma protuberans in cases where fresh or frozen samples of tumour tissue are not available.

Adolescent↗

PubMed1987

DNA cytofluorometry in micro-dissected paraffin embedded breast tissue: description of a method.

DNA microfluorometry on smears obtained from paraffin embedded tissue has been shown to be a distinctive possibility. In this paper a simple method for the detachment of cells from mammary ducts and ductules is described. Areas of interest were selected in conventional slides stained with Haematoxylin and Eosin. The corresponding paraffin embedded block was then dewaxed. The areas under study were retraced with a stereomicroscope and the cells within ducts and ductules were scraped out with a 0.4 mm diameter fine needle. Cells were isolated with mechanical and enzymatic procedures and stained with the Feulgen reaction. The DNA content of single cells was then measured using a microfluorometer.

Breast Neoplasms↗

PubMed1998

Immunohistochemical analysis of mycosis fungoides on paraffin-embedded tissue sections.

Mycosis fungoides (MF) is typically characterized by dermal and epidermal infiltration of T lymphocytes with a helper/inducer phenotype. Immunophenotypic analysis of such cases was traditionally performed by flow cytometry or immunohistochemistry on cryostat sections. With the advent of new monoclonal antibodies developed against T-cell antigens, including CD3, CD4, CD5, and CD8, it is now possible to immunophenotype T-cell subpopulations in paraffin-embedded tissues. To investigate the potential use of these antibodies for the evaluation of cutaneous lesions, 35 specimens (34 skin and 1 lymph node) from 29 patients with MF were retrospectively reviewed and immunophenotyped in paraffin sections with antibodies to CD3 (T-cell CD3), CD4 (NCL-CD4-1F6), CD5 (NCL-CD5-4C7), CD8 (CD8/144B), and CD20 (L26). Epidermal and dermal distribution of T and B cells were analyzed, and we assessed the ratios of CD4+ to CD8+ T cells. All of our 35 cases demonstrated a predominant CD3+ T-cell population. In 32 cases, the neoplastic cells expressed CD3, CD4, and CD5 consistent with a T-helper/inducer phenotype. In three cutaneous cases, the neoplastic CD4+ T cells showed minimal or absent expression of CD5, indicating an aberrant phenotype. In the majority of cases, minimal CD8+ T cells were present in the background, but in four cases, the CD4:CD8 ratios were 2:1 or less. Thirty-two cutaneous cases demonstrated epidermotropism exclusively by CD4+ T cells; one case showed both CD4+ and CD8+ T cells. In 17 cutaneous cases, scattered dermal CD20+ B cells were found individually or in small clusters within the background surrounding the neoplastic infiltrates. We concluded, therefore, that the immunophenotypic analysis of T-cell subpopulations using monoclonal antibodies of CD3, CD4, CD5, and CD8 was useful for histologic evaluation and confirmation of MF lesions in paraffin-embedded tissue. These antibodies might also provide an effective method of immunophenotyping other neoplastic and non-neoplastic T-cell populations in paraffin-embedded tissues.

Adult↗

PubMed1996

The use of microwave irradiation as a pretreatment to in situ hybridization for the detection of measles virus and chicken anaemia virus in formalin-fixed paraffin-embedded tissue.

Microwave irradiation was investigated as a pretreatment to in situ hybridization on formalin-fixed, paraffin-embedded tissue. Two probe/tissue systems were used: a single-stranded RNA probe for the detection of measles virus nucleocapsid genome in subacute sclerosing panencephalitis brain tissue, and a double stranded DNA probe for chicken anaemia virus in thymus of chicken infected with the virus. Microwaving, when used as sole pretreatment, was not as effective as the more traditional enzyme pretreatments for in situ hybridization. However, when used in combination with existing pretreatments, a significant increase was found in hybridization signal in both brain and thymus tissue. This was emphasized when combination enzyme/microwave pretreatments were used prior to detection of measles virus by in situ hybridization in a series of five archival subacute sclerosing panencephalitis cases. The use of microwave irradiation would be recommended as a means of supplementing in situ hybridization methods, especially when using long-term formalin fixed paraffin-embedded tissue.

Animals↗