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At least 361 records · Page 20Linked to original sources
PubMed2001

A technical note on microsatellite DNA instability studied in archival paraffin-embedded tissues.

The authors present brief overview of problems associated with analysis of microsatellite sequences in DNA from archival paraffin-embedded tissue. This methodology can serve several diagnostic and research purposes provided appropriate precautions are taken to extract DNA of acceptable quality with small admixture of contaminants. Also PCR amplification step requires some modifications compared to routine reactions involving DNA from fresh or freshly-frozen tissues.

DNA↗
PubMed2001

Immunofluorescence technique for 100-nm-thick semithin sections of Epon-embedded tissues.

An immunofluorescence staining method for Epon-embedded materials is described. Rat kidney and liver were fixed by perfusion with 1% glutaraldehyde for 10 min. Tissue slices were embedded in Epon. Semithin sections with thicknesses ranging from 1,000 to 100 nm were cut and mounted on clean glass slides. Epoxy resin was removed by treatment with 10% sodium ethoxide. Sections were digested with 0.05% trypsin and then treated with sodium borohydride. Sections were immunostained for leucine aminopeptidase (plasma membrane), catalase (peroxisomes), 3-ketoacyl-CoA thiolase (mitochondria), cathepsin D (lysosomes), and LGP107 (lysosomal membrane) using Cy(2)- or Alexa 546-labeled secondary antibodies. In 1,000-nm-thick sections, non-specific fluorescence remained and such fluorescence decreased as the sections became thinner. Clear specific fluorescence was obtained in the sections with thicknesses ranging from 250 to 100 nm with Alexa 546-labeled antibody (red fluorescence) but was not specific enough with Cy(2)- or Alexa 430-labeled antibody (green fluorescence). Sodium borohydride greatly abolished autofluorescence of glutaraldehyde. The present method made it possible to obtain signals in cross-sections of biological materials with a thickness of 250-100 nm, which are difficult to obtain in optical section using a confocal laser microscope.

Acetyl-CoA C-Acyltransferase↗
PubMed1995

Immunohistochemical detection of epidermal growth factor in glycol methacrylate embedded tissue.

This study addresses a variety of immunohistochemical conditions for detecting EGF in 3.5% paraformaldehyde fixed, glycol methacrylate embedded tissue including antigen unmasking with trypsin, dilution of primary antibody, and incubation time with primary antibody. Color development was achieved with a biotinylated secondary antibody linked to an avidin biotinylated peroxidase complex. Trypsinization and a 12 hr incubation with the primary antibody was essential to detect EGF in this system. Adequate staining could be achieved with a 1:100 dilution of the primary antibody.

Animals↗
PubMed2000

Intratumor heterogeneity of centromere numerical abnormality in multiple primary gastric cancers: application of fluorescence in situ hybridization with intermittent microwave irradiation on paraffin-embedded tissue.

Our recent success in retrieving distinct fluorescence signals in response to centromere specific probing of paraffin-embedded tissues after intermittent microwave (MW) treatment provided the opportunity to analyze chromosome numbers or centromere abnormality in situ in human tumors in various clinicopathological settings. In this study, centromere numerical abnormality (CNA) was investigated by fluorescence in situ hybridization (FISH) in a case of multiple gastric cancer having intratumor histological heterogeneity. The different profiles as determined using a total of 20 specific probes on 4 multifocal lesions in the stomach confirmed the multi-clonality of these tumors. FISH with probes specific for chromosomes 10, 11, 16 and 18 revealed intratumor heterogeneity of the CNA, which corresponded to the histological heterogeneity. Our report clearly demonstrates, for the first time, intratumor heterogeneity of CNA and its association with the histological picture, and substantiates the applicability of the MW-assisted FISH protocol to paraffin-embedded pathological specimens.

Centromere↗
PubMed2006

Simple salting-out method for DNA extraction from formalin-fixed, paraffin-embedded tissues.

The aim of this study was to standardize a method of DNA extraction from formalin-fixed and paraffin-embedded tissues (PETs) using a salt solution to precipitate protein and isopropanol to precipitate DNA. The samples were submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2 and 4M) were compared with a phenol-chloroform extraction method and with a commercial DNA isolation kit. DNA was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR. The 167 and 268bp fragments of APC and beta-globin genes, respectively, were amplified equally from DNA extracted by all tested methods and in all cases. However, the 536bp fragment of beta-globin gene was not amplified in all cases. According to our results, the extraction method using ammonium acetate proved to be simple and suitable for obtaining DNA of good quality, which can be easily amplified by PCR.

Acetates↗
PubMed2003

Detection of fusion gene transcripts in fresh-frozen and formalin-fixed paraffin-embedded tissue sections of soft-tissue sarcomas after laser capture microdissection and rt-PCR.

The diagnosis of small round cell sarcomas is often very difficult, especially when only small biopsy specimens are available for examination. Recent studies have shown that some sarcomas have specific recurrent chromosomal translocations producing chimeric gene fusions, which can be detected by reverse transcription-polymerase chain reaction (RT-PCR), fluorescent in situ hybridization (FISH), or cytogenetic analysis. In this study, 12 cases of well-defined sarcomas including Ewings sarcoma/primitive neuroectodermal tumors (ES/PNET), synovial sarcoma (SS), alveolar rhabdomyosarcoma (ARMS), and desmoplastic small round cell tumors (DSRCT) were used to collect specific numbers of cells by laser capture microdissection (LCM), subsequently used for RT-PCR to detect specific chimeric gene transcripts. Tumor cells from fresh-frozen (FS) tissue sections and paraffin-embedded (PS) tissue sections from the same cases were compared directly to evaluate the sensitivity of FS and PS sections as the starting material for analysis. Samples were used for RNA extraction, RT-PCR analysis, and Southern hybridization with fluorescein-labeled internal probes followed by enhance chemiluminescence (ECL) detection. The fusion gene transcripts could be detected using 50 cells from FS materials in all cases and from 1 cell in 9 of 12 cases. For PS, a positive signal could be detected using 200 to 1000 cells in all cases, while weaker signals were detected using 50 cells in most cases. These results indicate that the fusion gene products from small round cell sarcomas can be detected by RT-PCR with 10 to 200 cells from FS and PS tissues. The sensitivity of RT-PCR with FS was 10- to 50-fold greater than with PS. These results also suggest that RT-PCR analysis for sarcoma fusion gene products can be successfully performed when only a few cells are available for analysis, although this is not recommended for routine clinical use.

Blotting, Southern↗
PubMed1986

Diagnosis of the type of amyloid in paraffin wax embedded tissue sections using antisera against human and animal amyloid proteins.

Different histochemical techniques were compared on paraffin wax embedded tissue sections for routine classification of amyloid; the following methods were used: potassium permanganate, the indirect immunoperoxidase method using polyclonal anti-human amyloid antisera (anti-AA, anti-A lambda, anti-A kappa and anti-AF) and the peroxidase-antiperoxidase (PAP) method using antisera against human, bovine, hamster and canine AA amyloid. Anti-human AA antiserum appeared to be a useful tool in this respect. Polyclonal anti-AL antisera may be helpful in diagnosing AL amyloid, but were less of value than anti-AA serum. Strong cross reactivity between anti-bovine AA antiserum and human AA amyloid deposits was found. This indicates that animal amyloid AA antisera can also be used for the diagnosis of AA amyloid in human tissues.

Adult↗
PubMed1989

Phenotypic analysis of infiltrating cells in human myocarditis. An immunohistochemical study in paraffin-embedded tissue.

To define the cellular subpopulations that infiltrate the heart in human myocarditis, formaldehyde-fixed, paraffin-embedded sections from 18 hearts with histologically proved myocarditis were examined immunohistochemically. Disease was classified on routine stains as follows: mixed mononuclear cell (7 cases), granulomatous (3), giant cell (1), rheumatic (2), and fungal (5) myocarditis, respectively. On immunohistochemical examination, T-lymphocyte and macrophage predominance was found in nearly every case, except in fungal myocarditis, in which polymorphonuclear leukocytes and macrophages prevailed. In contrast, B lymphocytes and natural killer cells were conspicuously absent, regardless of histologic classification. Giant cells in giant cell myocarditis and in the Aschoff lesions of rheumatic carditis expressed macrophage, but not myocyte, antigens, suggesting derivation along macrophage lineage. Immunohistochemical data obtainable in paraffin-embedded tissues supplement the study of myocarditis, providing information potentially relevant to immunopathogenesis, natural history, and therapy.

Cell Line↗
PubMed1991

Removal of inhibitor(s) of the polymerase chain reaction from formalin fixed, paraffin wax embedded tissues.

A problem associated with use of the polymerase chain reaction to amplify specific DNA fragments from formalin fixed, paraffin wax embedded tissues is the not infrequent failure of amplification. One possible reason for this could be the presence of inhibitor(s), which interfere with the activity of the reaction. It has been shown that such inhibitor(s) exist when amplifying the human beta globin gene (which exists in human genomic DNA as a single copy gene) from routine clinical samples. A variety of methods to remove such inhibitor(s) were investigated. The results indicate that inhibitor(s) are removed by proteinase K digestion, followed by purification with phenol/chloroform, and centrifugation through a Centricon-30 membrane (30,000 molecular weight cut off). Other factors, including the length and concentration of the DNA sequence to be amplified, can also affect amplification.

Base Sequence↗
PubMed1995

Jacalin, another marker for histiocytes in paraffin-embedded tissues.

Jacalin is a lectin which reacts with D-galactose. We have tested jacalin on 75 samples of different formalin- and alcohol-fixed tissues. A consistent cytoplasmic stain of the histiocytes was observed in paraffin-embedded tissues in all cases studied of reactive sinus histiocytosis, macrophages in clear centres of follicular hyperplasia, in tuberculosis granulomas and in osteoclast-like giant cells in a breast carcinoma. We failed to find any clear binding of jacalin to the cells of eosinophilic granulomas, giant cell tumors of tendon sheath, pleomorphic malignant fibrous histiocytomas, Hodgkin's disease, melanomas, nevi or signet ring cell carcinomas of the breast and stomach. It seems that jacalin is a good marker for free histiocytes/macrophages, not for fixed histiocytes and tumors related to them. This lectin might play a role in differential diagnosis with histiocyte mimicking processes.

Breast Neoplasms↗
PubMed1998

Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections.

We describe and discuss a method of protein extraction for Western blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm2 50-micron-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernible protein bands ranging from 10 kD to 120 kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as beta-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.

Adenoma↗
PubMed1996

Detection of t(11;22)(q24;q12) translocation breakpoint in paraffin-embedded tissue of the Ewing's sarcoma family by nested reverse transcription-polymerase chain reaction.

Tumors of the Ewing's sarcoma family often present a major diagnostic challenge for the pathologist. In recent years, significant progress has been made in identifying characteristic chromosomal rearrangements associated with certain solid tumors. More than 85% of Ewing's sarcoma and related tumors present a specific t(11;22) (q24;q12) balanced translocation, which generates a fusion transcript of the EWS gene and the FLI-1 gene. The cloning of the t(11;22)(q24;q12) breakpoint has raised the possibility of using a reverse transcription-polymerase chain reaction (RT-PCR) based assay as a diagnostic tool. We report an improvement of the established method, which currently depends on fresh or snap-frozen tissue, so that it is possible to use formalin-fixed, paraffin-embedded tissue as a source of RNA. The described nested RT-PCR assay enables the pathologist to investigate retrospectively archival tumor samples or to confirm the diagnosis in cases where no fresh or frozen material is available.

Adult↗
PubMed1994

Quantitative analysis of chromosome in situ hybridization signal in paraffin-embedded tissue sections.

Interphase cytogenetic analysis using chromosome-specific probes is increasingly being used to detect chromosomal aberrations on paraffin-embedded tissue sections. However, quantitative analysis of the hybridization signal is confounded by the nuclear slicing that occurs during sectioning. To determine the sensitivity and accuracy of chromosome in situ hybridization for detecting numerical chromosomal aberrations on paraffin-embedded sections, in situ hybridization was performed on sections derived from mixtures of cell populations with known frequencies of numerical chromosomal aberrations and the Chromosome Index (CI) was calculated (i.e., total number of signal spots/number of nuclei counted) as a quantitative measure of chromosome copy number. The presence of 25% or more monosomic or tetrasomic cells in a given population was easily detected as a change in CI (P < 0.05). Lower degrees of polysomy could be detected as a small percentage of nuclear fragments with > 2 signal spots. The CI was not significantly influenced by a change in section thickness from 4 to 8 microM, by an increase in cell size from 478 to 986 microM3, or by the choice of detection method (fluorescence vs. conventional bright-field microscopy). Comparative analysis of touch preparations and tissue sections from the corresponding breast tumors showed that CI accurately reflects the average copy number of chromosomes in intact nuclei and may actually be superior to in situ hybridization on whole nuclei for the detection of numerical chromosomal changes in defined histologic areas. This method is thus a sensitive and accurate means of studying genetic changes in premalignant and malignant tissue, and of assessing the genetic changes associated with specific phenotypes.

Aneuploidy↗
PubMed1995

In situ hybridization for vasopressin mRNA in the human supraoptic and paraventricular nucleus; quantitative aspects of formalin-fixed paraffin-embedded tissue sections as compared to cryostat sections.

In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.

Aged↗
PubMed1991

An efficient method for the extraction of DNA from formalin-fixed, paraffin-embedded tissue by sonication.

A method was developed for fast and efficient isolation of DNA from formalin-fixed, paraffin-embedded tissue sections for subsequent use in PCRs and DNA hybridization assays. The method relies on the use of a sonicating water bath to disrupt tissue samples to which a small amount of micro-sized glass beads have been added. The sonicating glass beads provide fast and efficient physical shearing of fixed tissue sections, allowing for quick release and solubilization of the DNA. The extraction process from paraffin section to amplifiable target DNA takes 30 minutes. The method eliminates the need for repetitive solvent extractions and exhaustive proteinase K digestion. PCR amplification of human genomic and viral target sequences was successfully carried out on DNA isolated from a number of different types of normal and infected tissues.

Base Sequence↗
PubMed2001

Detection of gp43 and ITS1-5.8S-ITS2 ribosomal RNA genes of Paracoccidioides brasiliensis in paraffin-embedded tissue.

Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. An increase in PCM has been reported in recent years and the disease is now recognized as one of the imported fungal infections in Japan. To date, more than 15 cases of PCM have been reported in our country, and five of them were diagnosed by clinical and histopathological findings without mycological study. We applied 2 nested polymerase chain reaction (PCR) amplification methods for detecting P. brasiliensis genes from paraffin-embedded tissue specimens. Successfully amplified were: a 473 base pairs fragment of gp43 gene of P. brasiliensis (located from 741st to 1,213rd base), and a 418 base pairs fragment of 5.8S ribosomal RNA gene of P. brasilienisis which included internal transcribed spacers (ITS) 1 and 2 (located from 131st at ITS1 to 195th at ITS2) in paraffin-embedded murine tissues infected with P. brasiliensis yeast cells. The authenticity of the PCR products was confirmed by nucleotide sequence analysis. These results indicate that the two nested PCR methods may be useful for diagnosis of PCM.

Animals↗
PubMed1986

Effect of section thickness on quality of flow cytometric DNA content determinations in paraffin-embedded tissues.

DNA content determinations were carried out by flow cytometry on nuclear suspensions prepared from the same paraffin-embedded tissue block for each of eight surgically resected human carcinomas at section thicknesses of 5,10,20,30,40,50, and 100 millimicrons. Flow cytometric DNA determinations were also obtained on fresh tissue specimens in four of the eight carcinomas. As section thickness decreased below 50 millimicrons, there was a progressive increase in the histogram baseline noise at low DNA values and a decrease in the relative peak height of aneuploid DNA. The former was attributed to an increase of nuclear fragments in thinner sections, and the latter to the greater probability of transection of the larger aneuploid cells within a specimen. Both artifacts were minimized at section thickness of 50 millimicrons or greater.

Breast Neoplasms↗