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PubMed2003

[Application of DNA extraction and analysis of its influence factors].

To explore influence factors of DNA extraction, the protease and phenol-chloroform method was used to extract DNA in whole blood, fresh tissues, frozen tissues embedding with OCT and tissues embedding in paraffin. It results that 200-300 microg DNA was extracted from 1 ml whole blood or 0.2 g fresh tissues or frozen tissue embedding with OCT. DNA extracted from paraffin specimen can be directly used in PCR amplification. Purity DNA can be extracted from four kinds of different tissues. OCT hasn't harmful effect on tissue DNA extraction.

English Abstract↗
PubMed1991

Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue.

We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.

Animals↗
PubMed1991

A simple method for isolation of DNA from formalin-fixed paraffin-embedded samples for PCR.

A simple method of processing formalin-fixed, paraffin-embedded tissue sections for DNA amplification by polymerase chain reaction (PCR) is described. In this procedure, deparaffinized sections are readily subjected to DNA isolation simply by boiling and the released DNA can be directly employed for PCR. The method allows analysis of single-copy genes or viral sequences at least up to 300 base pairs long in one working day. This method is particularly useful in analysing retrospective materials when the simplicity and low cost of the assay are preferable. Furthermore, the simplicity of the procedure reduces the risk of contamination.

Base Sequence↗
PubMed1995

Mechanism for antigen detection on deplasticized epoxy sections.

The purpose of this investigation was to explain why deplasticizing of epoxy sections gives higher immunogold labeling than non-deplasticizing. The methods used were the following: (1) Comparison of the ratio of immunogold labeling of deplasticized and non-deplasticized sections with gold particles of different sizes and comparison of this ratio with respect to sections of different thickness, (2) the tilt method (Brorson et al., 1994). Human kidney tissue with amyloid A depositions, human fibrin, and human pituitary tissue were embedded, sections were deplasticized on grids, treated with anti-Aa, anti-fibrinogen or anti-ACTH (ACTH = adrenocorticotropic hormone), and reembedded on grids. Indications of significant antibody penetration were found only at the periphery of structures (ACTH-vesicles). This penetration was about 30 nm. The ratios of immunogold labeling of deplasticized and non-deplasticized sections were approximately 2, 5 and 1 for amyloid, fibrin and ACTH, respectively, and were independent of the gold particle size. No significant differences of gold labeling were found between thicker and thinner deplasticized epoxy sections regardless the gold particle size. No significant differences of gold labeling between deplasticized epoxy sections and LR-White sections were found on interior areas of ACTH-vesicles or amyloid A plaques. The increased labeling of deplasticized epoxy sections compared to normal epoxy sections seemed to be mainly a surface phenomenon. The practical significance of this observation is that deplasticizing of epoxy sections may be a better method for localizing antigens at the periphery of structures than the use of other resin embedding media. Deplasticizing of epoxy sections may be a method of choice in a pathological laboratory to detect antigens in routinely embedded tissues.

Adrenocorticotropic Hormone↗
PubMed1996

The Agar sandwich technique for retinal biopsy processing.

BACKGROUND: The authors developed an agar sandwich technique for retinal biopsy processing. This tissue agar embedding technique allows for a rapid and reliable method to handle and transport retinal biopsies from the operative field to the histology laboratory. METHODS: Biopsies from rabbit retinas infected with herpes simplex virus, epiretinal membranes from patients with macular pucker, and retinas from patients with acute retinal necrosis were studied. Each retinal biopsy was fixed, mounted on an agar disc, and covered with liquid agar. Light microscopy, electron microscopy, immunocytochemistry, and polymerase chain reaction were employed on the agar-embedded tissue. RESULTS: The tissues remained mounted in the agar sandwich and maintained their orientation throughout the processing. The morphologic integrity, histologic characteristics, antigenic properties, and DNA quality all were preserved using the agar sandwich technique. CONCLUSION: The agar sandwich technique is an efficient and simple technique for handling small biopsy specimens that require various analyses.

Agar↗
PubMed1995

Rapid primary microwave-aldehyde and microwave-osmium fixation: improved detection of rat parotid acinar cell secretory granule alpha-amylase using a post-embedding immunogold ultrastructural morphometric analysis.

Studies of methods for improved fixation are becoming increasingly important in the field of quantitative immunocytochemistry. We used microwave (MW)-assisted chemical fixation to show improved retention of salivary gland acinar cell secretory granule alpha-amylase detected by a quantitative immunogold method. Blocks (4-mm3) of rat parotid gland were fixed by the following methods: (a) MW irradiation in an aldehyde fixative (AF) for 6 sec; (b) immersion in AF for 1.5 hr; (c) MW irradiation in osmium tetroxide (OT) for 9 sec; (d) immersion in OT for 1.5 hr; or (e) Sequential MW AF, 10 sec, MW OT rapid treatment (SMAORT), 10 sec. Specimens were processed routinely for transmission electron microscopy. Thin sections of Epon-embedded tissues were exposed first to rabbit IgG anti-human salivary alpha-amylase and second to gold-conjugated goat anti-rabbit IgG. Granule area was obtained by a point counting method. Labeling density was calculated as the number of gold particles/micron 2 +/- SD. Specimens fixed in seconds by MW-AF, MW-OT, or SMAORT showed ultrastructural preservation similar to immersion fixation in AF or OT for 1.5 hr. Immunogold labeling density of granule alpha-amylase was highest for SMAORT (874 microns 2) compared to MW-AF (295 microns 2), MW-OT (248 microns 2), routine sequential immersion in AF and OT (229 microns 2), or immersion in OT (no aldehyde) (190 microns 2). This study establishes the improved retention of salivary gland acinar cell secretory granule alpha-amylase and markedly enhanced fixation speed for ultrastructural studies made possible by MW-chemical fixation protocols that use aldehydes and osmium.

Animals↗
PubMed2002

P53 protein expression in oral squamous cell cancer in relation to some of its clinicopathological variables.

Oral squamous cell cancer develops through a multistep process by the accumulation of genetic and phenotypic changes. Loss of P53 tumor suppressor gene function represents the most common genetic lesion in human cancer. The significance of P53 expression for the development and progression of oral squamous cell cancer has still to be evaluated. The aim of this study was to estimate relationships between P53 protein expression and some clinicopathological variables of established or presumed prognostic value. A series of 129 oral squamous cell cancers was investgated retrospectively for expression of P53 protein by immunohistochemistry of paraffin-embedded tissue sections. The slides were stained with H+E and by immunohistochemistry with anti-human P53 antibody. Positive immunohistochemical staining for P53 protein was present in 75 (58%) oral cancer cases. There were no statistically significant correlations between oral cancer P53 expression and tumor site, grading, mitotic index, invasive margin type, as well as patients age and sex. Our results suggest that immunohistochemical overexpression of P53 is an important markerof accomplished neoplastic transformation in oral cavity lesions but it does not play a crucial role in the tumor progression.

Adult↗
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↗
PubMed1987

gamma-Glutamyl transpeptidase demonstrated in tissue sections embedded in glycol methacrylate resin.

A method is described for the histochemical demonstration of gamma-glutamyl transpeptidase in tissue sections embedded in glycol methacrylate at low temperature. Enzyme activity was preserved by a short (3 h) fixation of tissue in 4% paraformaldehyde at 4 degrees C prior to embedding at 4 degrees C. Tissue embedded in glycol methacrylate combined good morphology with accurate enzyme localization. Blocks of the embedded tissue could be stored at room temperature for at least 3 months without loss of enzyme activity. The resin is non-fluorescent, allowing the use of the fluorescent coupling agent 5-nitrosalicylaldehyde to visualize the reaction product.

Acrylates↗
PubMed2004

RT-PCR analysis of RNA extracted from Bouin-fixed and paraffin-embedded lymphoid tissues.

In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]- and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA. To explore the possibility for giving accurate real time quantitative RT-PCR results, cDNA obtained from matched frozen, Bouin-fixed and formalin-fixed neoplastic samples (two diffuse large cell lymphomas, one plasmacytoma) was tested for the following target genes: CD40, Aquaporin-3, BLIMP1, IRF4, Syndecan-1. Delta threshold cycle (DeltaC(T)) values for Bouin-fixed and formalin-fixed paraffin-embedded tissues and their correlation with those for frozen samples showed an extremely high correlation (r > 0.90) for all of the tested genes. These results show that the method of RNA extraction we propose is suitable for giving accurate real time quantitative RT-PCR results.

Acetic Acid↗
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↗
PubMedDate not supplied

Antibodies that label paraffin-embedded mouse tissues: a collaborative endeavor.

Histology and immunohistochemistry are important tools in the study of human diseases and their respective animal models. The study of mouse models has been hampered by the absence of a large set of mouse-specific antibodies adapted to paraffin-embedded tissues. A total of 196 antibodies were tested on paraffin-embedded mouse tissues preserved in five different fixatives (Fekete's acid-alcohol-formalin, 10% neutral buffered formalin, 4% paraformaldehyde, IHC Zinc Fixative, and Bouin's fixative). The antibodies were targeted to proteins of the cytoplasm (n = 100), plasma membrane (n = 48), nucleus (n = 36), extracellular compartment (n = 5), cytoplasm/cell membrane (n = 4), and viral proteins (n = 3). A total of 83 antibodies provided an adequate signal to noise ratio. Of these, adequate labeling required heat-mediated epitope retrieval or enzymatic digestion for 32 and 8 antibodies, respectively. Epitope recognition was best for tissues fixed with Fekete's acid-alcohol-formalin. However, some proteins could be detected only in IHC Zinc Fixative, confirming that there is no single fixative suitable for the preservation of all epitopes. Four of 13 antibodies that failed to label their cellular targets on tissue sections successfully labeled whole-mount tissues, indicating that tissue processing plays an important role in epitope degradation. Regularly updated information on immunohistochemistry of normal and neoplastic mouse tissues is accessible online at (http://tumor.informatics.jax.org); links to antibody suppliers' web sites are provided.

Animals↗
PubMed2001

Frozen tumor tissue microarray technology for analysis of tumor RNA, DNA, and proteins.

Tissue microarray technology is a new method used to analyze several hundred tumor samples on a single slide allowing high throughput analysis of genes and proteins on a large cohort. The original methodology involves coring tissues from paraffin-embedded tissue donor blocks and placing them into a single paraffin block. One difficulty with paraffin-embedded tissue relates to antigenic changes in proteins and mRNA degradation induced by the fixation and embedding process. We have modified this technology by using frozen tissues embedded in OCT compound as donor samples and arraying the specimens into a recipient OCT block. Tumor tissue is not fixed before embedding, and sections from the array are evaluated without fixation or postfixed according to the appropriate methodology used to analyze a specific gene at the DNA, RNA, and/or protein levels. While paraffin tissue arrays can be problematic for immunohistochemistry and for RNA in situ hybridization analyses, this method allows optimal evaluation by each technique and uniform fixation across the array panel. We show OCT arrays work well for DNA, RNA, and protein analyses, and may have significant advantages over the original technology for the assessment of some genes and proteins by improving both qualitative and quantitative results.

Animals↗
PubMed2005

JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues.

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.

Adult↗
PubMed1995

Detection and sequence confirmation of Sin Nombre virus RNA in paraffin-embedded human tissues using one-step RT-PCR.

Sin Nombre virus (SNV) is the causative agent of hantavirus pulmonary syndrome (HPS). SNV RNA can be detected in fresh or frozen autopsy tissue samples by reverse transcriptase polymerase chain reaction (RT-PCR), and virus antigens can be identified by immunohistochemistry. A method was developed for demonstration of SNV RNA in formalin-fixed, paraffin-embedded tissues by RT-PCR. Virus genomes were detected in 8 of 12 (66.7%) fixed human tissue samples that were positive by immunohistochemistry, and RT-PCR prior to tissue fixation. By comparison with nucleotide sequences determined previously in fresh or frozen tissues of the same patients, identical genomic sequences were obtained, proving the authenticity of the PCR products. The study demonstrates that detection of SNV RNA in formalin-fixed, paraffin-embedded archival tissue by RT-PCR is feasible. This method allows retrospective studies on the phylogenetic epidemiology of SNV in North America.

Base Sequence↗
PubMedDate not supplied

High quality of DNA retrieved for Southern blot hybridization from microwave-fixed, paraffin-embedded liver tissues.

To overcome the degradation problem encountered in DNA extracted from formalin-fixed, paraffin-embedded tissue blocks, several methods of tissue fixation were examined in order to improve the quality of the DNA recovered for use in nucleic acid analysis. The fixation methods included formalin fixation alone, alcohol fixation alone, and microwave fixation with tissues immersed in phosphate-buffered saline (PBS), alcohol, or formalin. Unfixed fresh frozen tissue served as the control. Using hepatitis B virus (HBV) DNA sequences and the type I human procollagen gene as markers and liver tissue as a target, microwave fixation, with formalin omitted, not only preserved the DNA very well, but also the labile viral antigen. Both high molecular weight-integrated and free-form HBV DNAs were well preserved, and suitable for polymerase chain reaction and Southern blot analysis. The restriction enzyme fragment pattern of DNA recovered from these paraffin blocks was identical to that of unfixed fresh frozen tissue. Microwave fixation also preserved the labile preS2 epitope of the hepatitis B surface antigen (HBsAg) considerably better than formalin. These results suggest that microwave fixation is superior to routine formalin fixation for the preservation of excellent quality of genomic and viral DNAs for nucleic acid hybridization analysis. Alcohol, often used for nucleic acid purification, was also a good fixative for preserving DNA and the antigenicity of the labile antigen, especially when carried out in combination with microwave fixation.

Base Sequence↗
PubMed1996

Antibody NCL-CD5 fails to detect neoplastic CD5+ cells in paraffin sections.

Many low grade B-cell lymphomas, and most T-cells lymphomas, express CD5 on their surface. This expression has been demonstrated in fresh cells or frozen sections. Recently, a new monoclonal antibody to CD5, NCL-CD5, has been introduced that detects CD5 in paraffin-embedded tissues. Paraffin-embedded tissues from five patients with small lymphocytic lymphoma (SL), four patients with chronic lymphocytic leukemia (CLL), and one patient each with large granular lymphocytic leukemia, diffuse large cell lymphoma (T cell), and mycosis fungoides were stained with the NCL-CD5 antibody after unmasking the antibody with the steam/citrate technique. All these cases had CD5 positivity demonstrated by flow cytometry or on cytospin or frozen section preparations. In addition, one case of angiofollicular lymph node hyperplasia (CD5+), two cases of SL/CLL (CD5-), and one case CD5- T-cell lymphoma were also investigated. Of the 12 CD5+ malignancies, only 1, an SL, was positive with the NCL-CD5 antibody. In seven of these cases, both B-5 and formalin-fixed tissues were tested; the one positive case was positive only in the formalin tissue. The three CD5- malignancies were also negative in paraffin sections (P = .001; McNemar test). However, reactive T cells did stain in these biopsy sections. The case of Castleman's disease showed many CD5+ cells with the NCL-CD5 antibody. Although NCL-CD5 does indeed stain reactive T cells in paraffin sections, it does not appear to stain neoplastic CD5+ cells.

Antibodies, Monoclonal↗