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PubMed1986

Extraction of nuclei from selected regions in paraffin-embedded tissue.

A method for the extraction of nuclei from selected regions in paraffin-embedded tissue is described. Fifty-micrometer sections are cut, dewaxed, and rehydrated. For the final handling, the sections are manually transferred from one tray to another. The sections are put on a slide under a dissection microscope and the region of interest is isolated by scraping off the irrelevant region with a scalpel. An optimal number of single nuclei is obtained by incubation in a protease solution with intermediate syringing. The nuclei are washed and can be used for flow cytometry. Resuspension of the nuclei in foetal calf serum and cytocentrifugation results in preparations suited for image analysis. DNA cytometric measurements of nuclei in a carcinoma in situ and an invasive carcinoma region in breast tissue present in the same tissue block and in a severe dysplasia/carcinoma in situ (CIN III) region of the cervix are presented.

Breast Neoplasms↗
PubMed1991

Analysis of cell cycle kinetics using flow cytometry from paraffin-embedded tissues in endometrial adenocarcinoma.

Flow cytometric analysis of cell cycle kinetics from paraffin-embedded tissues was performed to evaluate the DNA heterogeneity and risk factor of endometrial adenocarcinoma. We studied 54 tumor samples and 6 controls with normal endometrium, and all samples were measured for S-phase fraction (SPF) and proliferative index (PI). Intratumor variations of SPF and PI values were evaluated at 3 different sites of the tumor; (1) primary endometrial lesion, (2) the site of myometrial invasion, and (3) metastatic lesions. SPF and PI values of endometrial adenocarcinoma were significantly higher than those of normal endometrium. There was a significant correlation between the PI value and the histological grade. SPF and PI values at the primary endometrial lesion had a significant correlation with the prognosis of this cancer at stage III or IV. SPF and PI values which were measured at the site of myometrial invasion tended to be lower than those from other sites. Our results indicate that the SPF and PI values of endometrial lesions are one of the important prognostic factors of endometrial adenocarcinoma at stage III or IV. In addition, DNA syntheses of endometrial adenocarcinoma cells display intratumor heterogeneity.

Adenocarcinoma↗
PubMed1982

Unconventional application of standard light and electron immunocytochemical analysis to aldehyde-fixed, araldite-embedded tissues.

A simple procedure for the immunocytochemical analysis of glutaraldehyde/formaldehyde-fixed, Araldite- or Epon-embedded tissues by either light or electron microscopy is presented. Retention of immunoreactive antigen in deplasticized sections was achieved by use of a low concentration of glutaraldehyde in the fixative in combination with a seldom-used plastic solvent. This protocol produced good ultrastructural preservation in tissues and large, high-quality, 2-micrometers thick, plastic-free sections. These semithin sections provided a level of structural and antigenic preservation, image resolution, and labeling intensity that surpassed all other conventional sectioning methods used for immunocytochemistry. The capacity to use a single tissue sample in studies designed for light and electron immunocytochemistry, in conjunction with existing autoradiographic and cytochemical techniques, makes this a very desirable method for routine tissue preparation in research and clinical applications.

Animals↗
PubMedDate not supplied

Efficient method for the proteomic analysis of fixed and embedded tissues.

Formalin-fixed and paraffin-embedded (FFPE) tissues present a particular challenge for proteomic analysis. Yet, most of the archived tissues in hospitals and tissue banks worldwide are only available in this form. We have developed conditions for removal of the embedding medium and protein digestion, such that informative tryptic peptides are released from fixed proteins which are suitable for analysis by liquid chromatography-mass spectrometry (LC-MS). We demonstrate that the peptide identifications made by this approach compare favorably to those made from matched fresh frozen tissue. Moreover, we demonstrate that a high level of sequence coverage can be observed for proteins of interest.

Amino Acid Sequence↗
PubMed1992

A simple method using PCR for direct sequencing of genomic DNA from frozen tumor tissue embedded in optimal cutting temperature compound.

Described here is a three-day protocol that directly yields DNA sequence after isolating and PCR amplifying genomic DNA from a small sample of frozen nasopharyngeal carcinoma tissue embedded in optimal cutting temperature (OCT) compound. The method is consistently successful, reproducible and will facilitate the rapid analysis of DNA sequence from very small samples.

Base Sequence↗
PubMed1994

Wet autoclave pretreatment improves the visualization of silver-stained nucleolar organizer-region-associated proteins in routinely formalin-fixed and paraffin-embedded tissues.

This study describes the effect of wet autoclave pretreatment of routinely formalin-fixed and paraffin-embedded tissues on silver-stained nucleolar organizer-region-associated proteins (AgNORs) staining. The treatment of sections at superheated steam temperature (120 degrees C) before silver incubation leads to a significantly better discrimination of single-interphase AgNORs. Counting "black dots" both by eye and by means of a computer-aided image-analyzing system revealed considerably higher AgNOR mean numbers per nucleus in all pretreated cases when compared with the respective untreated controls. However, mean AgNOR numbers increased after autoclave pretreatment in a nonlinear manner (pairwise comparison by means of Spearman's Rank correlation coefficient: rho = 0.5), whereas measurement of the AgNOR area was less influenced by autoclave pretreatment (rho = 0.8). It is concluded that wet autoclave pretreatment of routinely formalin-fixed and paraffin-embedded archival material should be applied before AgNOR staining, particularly when quantitation of AgNORs involves counting procedures.

Adenocarcinoma↗
PubMed1991

Immunohistologic separation of B-cell-positive granulomas from B-cell-negative granulomas in paraffin-embedded tissues with special reference to tumor-related sarcoid reactions.

Frozen and formalin-fixed paraffin-embedded tissue sections were studied concurrently in 17 cases of granulomatous lesions of different etiologies using antibodies recognizing either fixation-sensitive or fixation-resistant antigens. In fixed tissues, the antibodies 4KB5 and L26 for B cells and UCHL1 and MT1 for T cells gave results similar to those obtained in frozen tissues with anti-leu-12/leu-14 for B cells and T-3 for T cells. Paraffin-embedded sections from 35 additional cases of granulomatous lesions were studied retrospectively using the same markers. The combined results from all 52 cases show that granulomas can be divided into two main "families" according to the presence or absence of B cells within the granulomas: one is a B-cell-negative family of lesions to which sarcoidosis and mycobacterial infection belong; the other is a B-cell-positive family of lesions to which toxoplasmosis, granulomatous lesions of unknown significance and tumor-related sarcoid reactions belong.

Antibodies, Monoclonal↗
PubMed2003

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues.

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.

Animals↗
PubMed1994

Morphological detection of X- and Y-chromosomes in smears and paraffin-embedded tissues using a non-isotopic in situ hybridization technique (NISH).

Pharyngeal smears and paraffin-embedded tissue specimens (skeletal muscle, kidney) obtained from 10 male and 10 female individuals were evaluated using non-isotopic in situ hybridization (NISH) with commercial X- and Y-specific biotinylated probes which recognize the pericentromeric regions DXZ1 and DYZ1/DYZ3 of the X- and Y-chromosome, respectively. The results provide evidence that the morphological sex determination of a single cell can be performed by critical application of this staining method leading to one nuclear signal in "male" cells using the Y-specific probe whereas "female" cells are negative. In situ hybridization of "female" tissues with an X-specific probe results regularly in 2 signals whereas "male" cells show only one spot in the nucleus.

Adult↗
PubMed1982

Demonstration of monoclonal lymphoplasmacytic proliferations by immunofluorescence on routine formalin-fixed, paraffin-embedded tissue.

This study explores the use of immunofluorescence on routine formalin-fixed paraffin-embedded tissue to distinguish monoclonal from polyclonal lymphoplasmacytic proliferations. Sixteen tissues containing plasma cell or lymphocyte and plasma cell proliferations were studied. Five-micron sections were deparaffinized in xylene, rehydrated, and treated with 0.1% trypsin for two hours. After washing with phosphate-buffered saline, separate sections were stained with fluorescein-conjugated antibody to IgG, IgA, IgM, IgD, and IgE, and a single section was stained with both fluorescein-conjugated anti-kappa and rhodamine-conjugated antilambda. The latter section was useful to distinguish nonspecific adsorption of the fluorochromes. Where possible, results were correlated with immunoelectrophoretic studies of serum and urine. Eleven specimens with monoclonal and four specimens with polyclonal lymphoplasmacytic proliferations were readily identified, including a case of giant lymph node hyperplasia with a monoclonal IgDK plasma cell component (confirmed by specific absorption studies). Identification of monoclonality by immunofluorescence preceded immunoelectrophoretic identification in one case. One other case gave equivocal results by fluorescence. Further, the method worked on formalin-fixed decalcified tissues, although a somewhat heavier background staining was noted. This method offers a simple, reliable technique to establish or confirm the diagnosis of monoclonal lymphoplasmacytic lesions.

Adult↗
PubMed1981

Toxoplasma gondii infection of the central nervous system. Use of the peroxidase-antiperoxidase method to demonstrate toxoplasma in formalin fixed, paraffin embedded tissue sections.

The peroxidase-antiperoxidase immunohistochemical technique was employed to stain formalin fixed, paraffin embedded tissue sections from three cases of encephalitis caused by Toxoplasma gondii. We studied two cases of congenital infection and one case of acquired toxoplasmosis occurring in an immunocompromised host. The peroxidase-antiperoxidase method was exquisitely sensitive and highly specific and stained both the encysted and tachyzoite forms of the organism, as well as allowing for easy identification of infected cells. In two cases of necrotizing encephalitis--one congenital, the other acquired--widespread dissemination of the Toxoplasma organism throughout the neural parenchyma was visualized using the peroxidase-antiperoxidase stain. Brain biopsy material that had been obtained eight days prior to death in the case of adult acquired toxoplasmosis did not contain any of the characteristic tissue cysts and was not diagnostic for toxoplasmosis by conventional staining techniques. However, peroxidase-antiperoxidase staining of tissue sections from this biopsy unequivocally demonstrated both free tachyzoites and multiple infected cells. Further application of the peroxidase-antiperoxidase method should increase our understanding of the pathology and pathogenesis of toxoplasmic encephalitis as well as allowing timely diagnosis in cases presenting with neurologic symptomatology.

Animals↗
PubMed1988

Effect of trypsinization on the immunostaining of formalin-fixed, paraffin-embedded tissues.

We have investigated the action of trypsin treatment on formalin-fixed, paraffin-embedded tissue sections prior to immunostaining with a variety of commercially available poly- and monoclonal antibodies commonly used in surgical pathology. Although enzymatic predigestion often markedly reduced the background and enhanced the immunoreaction for certain antibodies, false negative staining or no enhancement of the staining was observed with other antibodies. We conclude that the indiscriminate practice of trypsinization in immunohistochemical procedures could be a significant cause of false negative results and of discrepancies between laboratories. Therefore, each laboratory must develop its own quality control program for variables such as enzymatic predigestion.

Antigen-Antibody Reactions↗
PubMed1990

Staining sections of water-miscible resins. 1. Effects of the molecular size of stain, and of resin cross-linking, on the staining of glycol methacrylate embedded tissues.

Penetration of hydrophilic acid and basic dyes into sections cut from glycol methacrylate (GMA)-embedded tissues was studied; as were the effects on such staining of superficial coatings of thin layers of GMA. Dye size was a major factor in controlling penetration of resin and staining of tissues. 'Large' dyes (greater than 1000 Da) entered GMA very slowly, and only stained those tissue components poorly infiltrated by resin. 'Small' dyes (less than 550 Da) penetrated GMA readily, and stained tissue components whether or not they were resin-infiltrated. Dyes of intermediate size penetrated the resin, but the staining of resin-infiltrated tissue elements was slow. Background staining of resin also varied with dye size. Large dyes gave no staining of GMA. Small dyes did, but were readily removed by water washing. Dye of intermediate size penetrated resin slowly, and once inside were lost slowly. This gave background staining which required use of the plasticizing solvent ethanol for its removal. Increases in resin cross-linking also reduced staining rates. As a consequence, it is possible to predict the probable suitability, or otherwise, of various staining reagents proposed for use with GMA sections; and also the probable influences of histoprocessing on stain penetration. In particular it is suggested that penetration of colloidal metals and macromolecular reagents (e.g. labelled antibodies and lectins) will be limited to resin-free structures, and to the surface of resin sections. The use of superficial GMA coatings as convenient semipermeable membranes for enzyme histochemistry is also noted.

Animals↗
PubMed2006

Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues.

BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold. RESULTS: We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC) tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH)-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb). When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar. CONCLUSION: Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.

Carcinoma, Non-Small-Cell Lung↗
PubMed2002

[Alterations of DPC4/SMAD4/MADH4 gene detected in paraffin-embedded tissues of human pancreatic carcinomas].

OBJECTIVE: To demonstrate the alterations of DPC4/SMAD4/MADH4 gene in paraffin-embedded tissues of pancreatic carcinomas. METHODS: Forty-six cases of resected specimens containing carcinomatous tissue and normal pancreatic tissue were analysed for possible DPC4 gene mutations by polymerase chain reaction (PCR)and single-strand conformation polymorphism (SSCP). The DNA sequencing technique was applied to determine the patterns of gene mutation in the PCR-SSCP positive cases. Fifty-six cases of pancreatic carcinoma along with the specimens corresponding normal pancreatic tissues were studied by in situ hybridization (ISH) and immunohistochemistry (IHC) techniques for gene expression in mRNA and protein level. RESULTS: The homozygous deletion rate of exon 1, 2, 3, 4, 8, 11 of DPC4 gene in pancreatic carcinoma was 28.26%, while the mutation rate of DPC4 gene was 21.74%. In these tumors, there were 3 cases of nonsense mutation, 5 cases of missense mutation, 1 case of deletion and missense mutation, 1 case of insertion mutation. Positive rates of SMAD4 in carcinomatous tissues detected by the ISH and IHC were 53.57% and 58.93% respectively, whereas they were 91.07% and 89.29% in the matched normal tissue respectively. There were significant difference between cancer and normal tissue (P < 0.05). Thrity-two cases were positive of DPC4/SMAD4 with all methods mentioned above, the coincident rate was 87.50% (28/32). The coincidence between gene detection and ISH of SMAD4 was 87.50%, and it was 96.88% between gene detection and IHC of SMAD4. Of all 56 cases, the coincidence of the positive rates of SMAD4 detected by ISH and IHC was 91.07%. No significant difference among the positive rates of DPC4/SMAD4 as detected by the three different techniques (P > 0.05). CONCLUSIONS: The main mechanisms of inactivation of DPC4 gene in pancreatic carcinoma are homozygous deletion and mutation. The product of DPC4 expression is significantly decreased in cancer group compared with the normal tissues. As a tumor suppressor gene, DPC4 alteration is an important molecular event in pancreatic carcinoma, and probably plays a crucial role in cancer development and progression.

Adult↗
PubMed1999

Distribution of sialic acid-dependent carbohydrate epitope in thyroid tumors: immunoreactivity of FB21 in paraffin-embedded tissue sections.

The reactivity of monoclonal antibody FB21, which recognizes a sialic acid-dependent carbohydrate epitope, was tested with 94 non-neoplastic and neoplastic thyroid tissue specimens using immunohistochemical methods on formalin-fixed, paraffin-embedded tissue sections. These specimens included 11 cases of adenomatous goiter, three cases of Basedow's disease, 30 cases of follicular adenoma, 20 cases of papillary carcinoma, 15 cases of follicular carcinoma, six cases of medullary carcinoma, and nine cases of anaplastic carcinoma. FB21 reacted with 14 of 15 cases of follicular carcinoma that showed a microfollicular or trabecular pattern, and with nine of 20 cases of papillary carcinoma. A positive reaction was found on the cell surface membranes or apical parts of neoplastic follicles. FB21 also reacted with five of 30 cases of follicular adenoma. These cases showed a follicular pattern and positive staining pattern similar to that in follicular carcinoma. Adenomatous goiters, Basedow's disease, medullary carcinomas, and anaplastic carcinomas were negative for FB21 reactivity. Although the different reactivities of FB21 with papillary carcinoma and follicular carcinoma remain to be investigated, the high frequency of reactivity with FB21 suggests that it may be useful as a complement to morphological diagnosis in follicular carcinoma.

Adenoma↗
PubMed2001

Comparison of the DNA extraction methods for polymerase chain reaction amplification from formalin-fixed and paraffin-embedded tissues.

To obtain an adequate quality and quantity of DNA from formalin-fixed and paraffin-embedded tissue, six different DNA extraction methods were compared. Four methods used deparaffinization by xylene followed by proteinase K digestion and phenol-chloroform extraction. The temperature of the different steps was changed to obtain higher yields and improved quality of extracted DNA. The remaining two methods used microwave heating for deparaffinization. The best DNA extraction method consisted of deparaffinization by microwave irradiation, protein digestion with proteinase K at 48 degrees C overnight, and no further purification steps. By this method, the highest DNA yield was obtained and the amplification of a 989-base pair beta-globin gene fragment was achieved. Furthermore, DNA extracted by means of this procedure from five gastric carcinomas was successfully used for single strand conformation polymorphism and direct sequencing assays of the beta-catenin gene. Because the microwave-based DNA extraction method presented here is simple, has a lower contamination risk, and results in a higher yield of DNA compared with the ordinary organic chemical reagent-based extraction method, it is considered applicable to various clinical and basic fields.

DNA Primers↗