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At least 1,225 records · Page 68Linked to original sources
PubMed2005

Fluorescent in situ hybridization (FISH) on paraffin-embedded placental tissues as an adjunct for understanding the etiology of early spontaneous abortion.

OBJECTIVES: An investigation of first-trimester spontaneous abortions (SAs) for those cases in which karyotype is not available was designed to test the efficiency of fluorescence in situ hybridization (FISH) on paraffin-embedded tissues combined with pathological examination for understanding the etiology of SAs. METHODS: Pathological examination of 202 placental tissues from SAs was performed. FISH analysis was then carried out on paraffin-embedded tissue sections from the same abortion products with probes specific for chromosomes 13, 16, 18, 21, X, Y. RESULTS: FISH could be achieved in 196 cases (97%). After pathological analysis alone, the etiology of SAs was evoked in 40 cases. The suspected diagnosis was confirmed by FISH in 26 cases (13.2%). After combined pathological and FISH analysis, the etiology of SAs was identified in 83 from the 196 cases (42.3%) with the probe set used. CONCLUSION: The present study demonstrates the value of FISH on paraffin-embedded tissues as an adjunct for understanding the etiology of SAs for those cases in which karyotype is not available. Combination of pathological and FISH analysis increases the yield of diagnosis by a factor of 3.2. The results also demonstrate that predictions of the karyotype from pathological examination should be avoided.

Abortion, Spontaneous↗
PubMed1984

Incident light microscopy of surfaces of plastic embedded hard tissues.

To eliminate sectioning artefacts, we devised a method for the preparation of surfaces of plastic embedded hard tissues and their examination by incident light fluorescence microscopy. Flat surfaces produced by cutting on a microtome or using a low-speed saw were stained with dyes in aqueous solutions. Best results were obtained in tissues embedded in glycol-methacrylate. This polymer allowed good penetration of the dyes and its optical properties render cutting defects invisible with the present method. The relationship between hard and soft tissues was well preserved and the integrity of the mineralized component maintained. Cellular details and the distinction between osteoid and bone could be clearly/demonstrated. The procedure is simple, rapid and produces good results even in instances when histological sectioning is unsatisfactory.

Animals↗
PubMed1994

Detection of hepatitis C viral sequences in formalin-fixed, paraffin-embedded liver tissue: effect of interferon alpha therapy.

To determine the effect of interferon-alpha (IFN) therapy on hepatitis C virus (HCV) in liver, reverse transcription "nested" polymerase chain reaction (RT-PCR) was applied to detect HCV RNA in formalin-fixed, paraffin-embedded liver biopsy specimens obtained before and at the end of IFN therapy in 42 patients with chronic HCV infection. Results were correlated with the clinical and biochemical outcome in 36 cases. Fifteen patients were nonresponders to IFN; 13 patients had a complete response to IFN but relapsed shortly after IFN was stopped (responders who relapsed); and 8 patients showed a complete and sustained response to IFN therapy (sustained responders). Total RNA was extracted using proteinase K digestion and phenol/chloroform/isoamyl alcohol extraction, and HCV RNA was detected by standard RT-PCR using primers from the highly conserved 5' untranslated region. HCV RNA was detected in 41 of the 42 pretreatment specimens. Of the 36 patients with paired posttreatment samples, HCV RNA was detected in all 15 patients who did not respond to IFN and 9 of 13 who responded to IFN but relapsed shortly after IFN was stopped. In contrast, only one of the eight patients who had a sustained response to IFN therapy had HCV RNA detected by RT-PCR (P < 0.04). These data confirm 1) the feasibility of detecting HCV RNA in formalin-fixed, paraffin-embedded tissue from patients with chronic HCV infection, 2) show that sustained response to IFN is associated with loss of liver HCV RNA at the end of IFN therapy, and 3) offer an explanation for recurrence in patients who relapse.

Base Sequence↗
PubMed1997

Hepatic regeneration in humans with various liver disease as assessed by Ki-67 staining of formalin-fixed paraffin-embedded liver tissue.

The ability to document nuclear proliferation in the liver is essential to our understanding of hepatic regeneration and hepatocellular carcinoma. While numerous tests are available to provide such information in experimental animals, few can be applied to patients with liver disease. Ki-67 is a proliferating nuclear antigen present in replicating cells. Recent data indicates that staining for Ki-67 reflects nuclear proliferation in these tissues. To date, the technique has had only limited application to human liver tissues in formalin-fixed paraffin embedded tissue. In the present study, we documented Ki-67 staining in archival liver tissue from patients with mild chronic hepatitis, severe chronic hepatitis, inactive cirrhosis, hepatocellular carcinoma, and in normal livers. We found that Ki-67 staining was increased in patients with mild chronic hepatitis (labelling index 29 +/- 25), severe chronic hepatitis (labelling index 41 +/- 40), and hepatocellular carcinoma (labelling index 71 +/- 61), when compared to patients with inactive cirrhosis, (labelling index 1.4 +/- 3.1), and normal livers (labelling index 2.5 +/- 3.2). In conclusion, Ki-67 maybe useful tool to assess hepatocyte proliferation in formalin-fixed paraffin-embedded human liver tissue.

Carcinoma, Hepatocellular↗
PubMed1985

Staining of different endocrine cells with hydrochloric acid-toluidine blue in Epon embedded rat tissue.

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 hr in 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.

Adrenal Medulla↗
PubMed1976

Utilization of acrylate-embedded large tissue samples in dermatohistology.

A method of embedding tissue in synthetic material which has seldom been used in dermatohistology is described in this communication. This process combines the preparative advantage of slight artifactual alteration of organic tissue and readily reproducible sections between 1 and 3 mu in thickness with the feature of light microscopy that tissue slices of "normal" dimensions can be prepared without difficulty. It avoids the disadvantages of the small dimensions of the preparations required for the ultramicroscope. With few exceptions, all staining methods applied to paraffin slices can also be used.

Histological Techniques↗
PubMed2003

PCR detection of Actinobacillus pleuropneumoniaeapxIV gene in formalin-fixed, paraffin-embedded lung tissues and comparison with in situ hybridization.

AIMS: Formalin-fixed, paraffin-embedded lung tissues from pigs experimentally infected with 12 Actinobacillus pleuropneumoniae serotypes were used to develop nested PCR for the detection of apxIV gene. METHODS AND RESULTS: The PCR results from formalin-fixed, paraffin-embedded tissues were compared with in situ hybridization. The apxIV gene was detected in formalin-fixed, paraffin-embedded lung tissues from all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes by nested PCR. In situ hybridization produced a distinct positive signal in all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes. Agreement rates between nested PCR and in situ hybridization were 100% for the detection of apxIV gene in formalin-fixed paraffin-embedded lung tissues. Acceptable PCR signals were detected from lung tissues fixed for periods up to 180 days. CONCLUSIONS: The apxIV gene is species-specific rather than serotype-specific and is therefore an important diagnostic marker. The nested PCR assay would be a useful method for the detection of apxIV gene to diagnose A. pleuropneumoniae infection when formalin-fixed tissues are submitted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirmed the possibility of using formalin-fixed, paraffin-embedded tissues for the diagnosis of A. pleuropneumoniae infection in pigs.

Actinobacillus pleuropneumoniae↗
PubMed1988

Immunohistochemical antigen demonstration in plastic-embedded lymphoid tissue.

We describe a method for post-embedding immunohistochemical demonstration of a wide range of antigens in glycol methacrylate-embedded tissue. Rat spleen and thymus tissues were fixed by immersion in fixatives containing different concentrations of paraformaldehyde, washed in sucrose phosphate buffer, dehydrated in acetone, infiltrated in a glycol methacrylate mixture in which the commonly used softener 2-butoxyethanol was replaced by butaandiol monoacrylate, and embedded. Trypsin was used to re-expose the masked antigenicity. Excellent results were obtained with a panel of monoclonal antibodies (MoAbs) directed against T-cells, B-cells, Ia-positive cells, macrophages, follicular dendritic cells, and leucocyte common antigen-bearing cells. The method described combines exact localization of antigens with optimal tissue morphology.

Acrylates↗
PubMed2005

New approaches for genotyping paraffin wax embedded breast tissue from patients with cancer: the Iowa women's health study.

BACKGROUND: The use of paraffin wax embedded tissue samples as a source of DNA for genotype analysis has been limited because of difficulties in DNA extraction and single nucleotide polymorphism (SNP) analysis. AIMS: To test the feasibility of applying the combination of a commonly used DNA isolation procedure, PureGene, and a high throughput SNP analysis method, the polymerase chain reaction (PCR)-INVADER assay, to genotype several types of paraffin wax embedded breast tissues. METHODS: Twenty formalin fixed, paraffin wax blocks were obtained from five participants in the Iowa women's health study. Each participant provided several types of tissue including normal lymph node, normal nipple/areola tissue, inflammatory/fibrotic breast tissue, or normal breast tissue, and tumour tissue. RESULTS: Good quality DNA (260/280 ratio >1.6) was obtained from all tissues. Normal lymph nodes yielded the largest amount of DNA (97.1 mug). DNA obtained from the samples was tested for a germline C1183T polymorphism in the MnSOD gene by three methods-PCR-RFLP (restriction fragment length polymorphism), INVADER assay, and PCR-INVADER assay. Of the 20 samples, PCR-RFLP genotyped 16, the PCR-INVADER assay 18, and the INVADER assay two. This methodology was then used to analyse five additional genotypes and confirmed the general applicability of the method. CONCLUSIONS: This study demonstrated the feasibility of (1) using several paraffin wax embedded breast tissues as a source of DNA for germline genetic analysis, with lymph nodes providing the highest yield, and (2) using the combination of a common extraction method with a high throughput SNP analysis method, the PCR-INVADER assay.

Breast Neoplasms↗
PubMed1991

Suggestions for the quantitative X-ray microanalysis of thin sections of frozen-dried and embedded biological tissues.

When a microregion in a thin section of frozen-dried and embedded tissue is analysed by the conventional electron-probe X-ray continuum-normalization method, the measured quantity is in mmol of element per kg of embedded specimen. As each microregion contains an unknown amount of embedding medium, this quantity generally lies indeterminately somewhere within the wide range between mmol of element per kg of hydrated tissue and mmol of element per kg of dehydrated tissue. However, if a 'tag' element is incorporated in the embedding medium, the contribution of the medium to the local continuum count in each probed field should be measureable, and the X-ray data may then unambiguously yield mmol of element per kg of dehydrated tissue. This result should not be affected by shrinkage on freeze-drying or by incomplete replacement of water by embedding medium. The same X-ray data can additionally provide estimates of mmol of element per unit volume, mmol of element per kg of hydrated tissue and local dry-mass fraction. However, these estimates are subject to errors due to tissue shrinkage, incomplete replacement of water and beam damage.

Animals↗
PubMed1983

An improved method for embedding hard tissue in polymethyl methacrylate.

An improved routine method for embedding tissue, especially hard tissue, in polymethyl methacrylate (pMMA) is described. The improvements were: the final dehydration step before MMA infiltration was performed with methanol in a Soxhlet apparatus; the stabilizer hydroquinone was not extracted from the monomer (MMA), and more important, the commonly used polymerization initiator, benzoyl peroxide (bpo), was replaced by the initiator, bis (4-tert-butylcyclohexyl)peroxydicarbonate (bbpd). Bbpd is preferred to bpo because it is not explosive, far less is needed and it has a suitable half life. Moreover, bbpd, as obtained from the manufacturer, needs no further purification, in contrast to bpo. Temperatures during bbpd initiated polymerization did not exceed 48 C. In bbpd initiated pMMA, bubbles were almost never generated.

Benzoyl Peroxide↗
PubMed1991

RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification.

Fixed and paraffin-embedded tissues from pathology department archives can be available for RNA expression analysis. In this report, we show that RNA isolated from surgical or autopsy tissues, routinely processed by fixation and paraffin embedding, is not completely degraded. RNA fragments around 100-200 bases in length are still present even in organs late fixed and very rich in RNase, such as the pancreas. Here we describe a general protocol to obtain RNA from single 6-8-microns tissue sections. The RNA extracted can be analyzed for the presence of specific sequences by reverse transcription and amplification with the PCR. We studied the retinoblastoma gene expression in 38 human pancreas specimens from surgical or autopsy origin.

Animals↗
PubMed1989

Modification of the silver impregnation technique of Bielschowsky for use in glycol methacrylate-embedded brain tissue.

The modified Bielschowsky stain has become the standard silver impregnation technique for use in paraffin-embedded tissue sections for identifying abnormalities of neuritic processes in a variety of neurodegenerative disorders such as Alzheimer's disease. Recently, glycol methacrylate embedding has been used increasingly as a substitute for paraffin embedding of tissue, because it results in less tissue distortion during processing and provides greater clarity and resolution of cellular architecture and structural details. We have been able to modify the Bielschowsky stain for use in glycol methacrylate-embedded brain sections. The use of this technique will permit more accurate quantitation of the histopathologic changes in neuritic processes in neurodegenerative diseases.

Acrylates↗
PubMed1994

Detection of T-cell receptor beta chain mRNA in frozen and paraffin-embedded biopsy tissue using digoxigenin-labelled oligonucleotide probes in situ.

In situ hybridization techniques using a cocktail of digoxigenin-labelled T-cell receptor (TcR) constant (C) region beta oligonucleotide probes were used to detect TcR beta mRNA in frozen and paraffin-embedded tissue sections. The specificity of the C beta cocktail was confirmed by Northern blot analysis. The TcR C beta cocktail successfully hybridized to T cells in frozen and paraffin-embedded tissue obtained from patients with inflammatory arthropathies, B- and T-cell non-Hodgkin's lymphoma (NHL), and reactive tonsillitis, and showed staining patterns comparable to those obtained by conventional immunohistological detection of T cells. This is the first report of in situ studies using labelled TcR C beta oligonucleotide probes and may indicate the feasibility of investigating clonal T-cell populations using digoxigenin-labelled clonospecific probes in clinical samples in situ.

Biopsy↗
PubMed1998

Monoclonal antibodies raised to paraffin wax embedded archival tissue; feasibility study of their potential to detect novel antigenic markers.

A study to determine the feasibility of using archival paraffin wax embedded tissue to generate monoclonal antibodies is described. Specifically, monoclonal antibodies were raised to paraffin wax embedded normal human kidney tissue to test the possibility of producing antibodies to such tissue samples prior to attempting generation of antibodies to valuable archival tissue. Multiple sections (10 x 5 microm) were pooled and dewaxed as for immunohistochemical procedures and combined with Freund's adjuvant for immunization of BALB/c mice in vivo. Immunized spleen cells were fused with SP2 myeloma cells and subsequent clones screened on paraffin wax embedded normal human kidney sections, a range of cell lines and normal mouse tissue. Supernatants from 11 wells (from a total of 90 wells screened) showed different staining patterns on sections of paraffin wax embedded kidney. One clone, 1/11C, (isotype IgG1) which exhibited strong staining on all kidney tubules by immunohistochemical studies (glomeruli interstitium and vessels were unstained) and identified a band at 52 kDa on immunoblots of dewaxed kidney tissue (as used for immunogen) was chosen for further characterization. Immunoblotting of five mammalian cell lines showed differential expression of this 52 kDa band (distinct expression on 3/5, weak expression on 2/5 cell lines) whereas, all cell lines displayed a band at 44 kDa and a third band at 70 kDa was observed on 2/5 cell lines. In mouse tissue extracts, the 52 kDa band was identified in kidney tissue only (not in the lung, liver or spleen) with the 44 kDa and 70 kDa bands weakly expressed in all tissues. This preliminary investigation of a novel approach to identifying possible new antigenic markers or producing monoclonal antibodies which react better to known antigens on sections of paraffin wax embedded tissue showed that this method is feasible. The need to have a comprehensive screening system in place and the ability to identify potentially useful clones after the initial screening is paramount due to the relative scarcity of screening material (archival tissue sections) and the tedious nature of the screening method.

Animals↗
PubMed1981

Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue.

The recently developed low temperature embedding procedure with the resin Lowicryl K4M (Carlemalm E, Garavito M, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 656; Garavito M, Carlemalm E, Villiger W: Proc 7th Eur Cong Electron Microsc, 1980, p 658) was tested for its suitability for embedding of glutaraldehyde-fixed rat pancreatic tissue and for postembedding staining of thin sections with the protein A-gold (pAg) technique (Roth J, Bendayan M, Orci L: J Histochem Cytochem 26:1074, 1978) for amylase. Compared to conventional Epon embedding of glutaraldehyde fixed tissue, the low temperature embedding method with Lowicryl K4M resulted in a superior preservation of the general cellular fine structure, particularly in the Golgi apparatus. For low temperature embedded tissue, the quantitative evaluation of the immunocytochemical labeling for amylase showed a more specific staining of the rough endoplasmic reticulum, the Golgi apparatus, and the zymogen granules. This was due to a significant lowering of the background staining over all cellular organelles. The use of Lowicryl K4M at low temperature, due to the superior preservation, yields improved resolution and specificity in immunocytochemical postembedding staining.

Animals↗
PubMed1993

Detection of bovine viral diarrhea virus RNA in formalin-fixed, paraffin-embedded brain tissue by nested polymerase chain reaction.

Isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissues is delicate due to its fragility and ubiquitous ribonucleases. For retrospective studies, however, a convenient procedure for the detection of RNA in archived material is of great value. Bovine viral diarrhea (BVD) virus is a member of the pestivirus genus in the family Flaviviridae. Different protocols for the isolation of BVD virus RNA from fresh and autolytic as well as from routinely formalin-fixed and paraffin-embedded brain tissue of BVDV-infected calves were compared. The polymerase chain reaction (PCR) after reverse transcription (RT-PCR) was carried out subsequently for the detection of viral RNA. Using proteinase K digestion of deparaffinized tissue sections without additional ribonuclease inhibitors and subsequent nested PCR, a 803 bp fragment of the gene coding for the nonstructural protein p125 of BVD virus could be consistently detected. In addition, BVD virus RNA was detected by RT-PCR from non-fixed brain tissue after 10 days of autolysis.

Animals↗
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↗