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Results for “Paraffin Embedding”

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PubMed1991

The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. III. Extraction and purification of RNA and application to slot-blot hybridization analysis.

RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.

Animals↗

PubMed1988

Immunohistochemical demonstration of estrogen receptors (ER) in formalin-fixed, paraffin-embedded human breast cancer tissue by use of a monoclonal antibody to ER.

We describe an immunohistochemical method using a monoclonal antibody to localize estrogen receptors (ER) in formalin-fixed, paraffin-embedded tissue. The avidin-biotin-peroxidase complex method was used, preceded by trypsin treatment to expose antigenic sites. In 111 breast cancer specimens studied simultaneously by a dextran-coated charcoal (DCC) assay and the paraffin section method, agreement on receptor status was found in 101 (91%) specimens. Quantitative staining features showed a high degree of correlation with the results of the steroid binding assay (r = 0.81). Studies on the influence of fixation on ER localization done in rabbit uteri showed that fixatives mainly composed of coagulating reagents (Carnoy's, Zenker's, Bouin's, Lilly's AAF, Helly's, ethanol) precluded ER staining, whereas cross-linking fixatives (formaldehyde, glutaraldehyde) preserved antigenic sites, although the immunoreactivity of the receptor was somewhat decreased. Studies on the effect of enzyme preincubation showed this to increase antigenic expression of ER in formaldehyde-fixed breast tumors and in formaldehyde-, glutaraldehyde-, and Zamboni-fixed rabbit uteri.

Animals↗

PubMed1985

Application of DNA flow cytometry to paraffin-embedded archival material for the study of aneuploidy and its clinical significance.

By using a recently developed flow cytometric method we have analyzed cellular DNA content of paraffin-embedded histological material from cancer patients. This method allows the retrospective study of tumors from patients whose clinical outcome is already known, and we have applied it to ovarian cancers, stage II breast cancers, and to metastatic adenocarcinoma of unknown primary site. In addition to knowledge of patient survival, comprehensive information was available about other prognostic determinants and treatment received, and we have used multivariate analysis in an attempt to determine the prognostic significance of cellular DNA content. In ovarian cancer, it is a major prognostic variable except in stage IV disease, whereas in metastatic adenocarcinoma of unknown primary site cellular DNA content has no influence on survival. For stage II breast cancer the situation is more complex and requires larger numbers to be studied. However, aneuploid tumors tend to have more extensive involvement of axillary lymph nodes and a poorer overall disease-free survival. This influence of DNA content on disease-free survival appears to be confined to premenopausal patients, and has no effect on patient survival following disease recurrence. Although we need to study more patients and more tumor types, taken together the results so far show a generally more favorable prognosis for patients with diploid tumors, except in the presence of recurrent or metastatic disease. The better prognosis associated with diploid tumors could be due to the fact that they are more commonly found in earlier clinical stages rather than to their being inherently less aggressive than aneuploid tumors.

Aneuploidy↗

PubMed1986

Are keratin proteins a better tumor marker than epithelial membrane antigen? A comparative immunohistochemical study of various paraffin-embedded neoplasms using monoclonal and polyclonal antibodies.

Epithelial membrane antigen and keratin proteins represent markers of epithelial differentiation that may be detected in routine formalin-fixed, paraffin-embedded tissues. Eighty-seven neoplasms, including 48 adenocarcinomas of various types, squamous and transitional cell carcinomas, small-cell anaplastic carcinomas, carcinoid tumors, mesotheliomas, hepatomas, melanomas (metastatic), adrenal cortical carcinomas, germ cell tumors, and extramammary Paget's disease, were assessed to determine the relative effectiveness of these antigens as tumor markers. Immunoperoxidase studies were performed using monoclonal antibodies to epithelial membrane antigen and monoclonal (combined AE1 and AE3) and polyclonal (bovine muzzle keratins) antibodies to keratin proteins. In more than half the cases (50/87%), both markers yielded comparable results. However, in 29 cases (33%), keratin proteins were clearly superior to epithelial membrane antigen as a tumor cell marker. Particular discrepancies were apparent for some gastrointestinal adenocarcinomas, squamous cell carcinomas, hepatomas (hepatocellular type), spindle cell components of mesotheliomas, and carcinoid tumors. Epithelial membrane antigen represented a better marker in eight cases (9%), mainly for small-cell anaplastic carcinomas and some renal cell and pulmonary adenocarcinomas. Adrenal cortical carcinomas, melanomas, and seminomas were nonimmunoreactive for both antigens. Epithelial membrane antigen and keratin proteins represent useful complementary markers in diagnostic surgical pathology.

Antibodies, Monoclonal↗

PubMed1994

Immunohistochemical identification of Renibacterium salmoninarum by monoclonal antibodies in paraffin-embedded tissues of Atlantic salmon (Salmo salar L.), using paired immunoenzyme and paired immunofluorescence techniques.

Renibacterium salmoninarum was identified in situ by immunoenzymatic and immunofluorescence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon (Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57-58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin-peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1979

Demonstration of alpha 1-antitrypsin by immunofluorescence on paraffin-embedded hepatic and pancreatic tissue.

Studies were performed in an attempt to improve current immunohistological techniques for the demonstration of alpha 1-antitrypsin (A1AT) in formalin-fixed paraffin-embedded tissues. The unwanted fluorescence (UF) commonly occurring in such procedures was found to be effectively eliminated by immunoadsorption of A1AT antisera with human serum lacking A1AT (Pi-null phenotype) coupled in solid phase to glutaraldehyde-activated aminohexyl-Sepharose 4B. Specificity of the antisera for A1AT was established by subsequent solid phase immunoadsorption against normal human serum bound to AH-Sepharose 4B. Using these techniques, immunoreactive A1AT was demonstrated in the cytoplasm of hepatocytes in liver biopsies obtained from patients with Z and MZ serum phenotypes, and in the cytoplasm of normal pancreatic islet cells.

Animals↗

PubMedDate not supplied

Detection of estrogen receptor in formalin-fixed and paraffin-embedded breast carcinoma: correlation with histological patterns.

The authors studied the expression of estrogen receptor (ER) in tissues of breast carcinomas which were previously fixed in formalin and paraffin-embedded. The ER expression was correlated with several histological findings, namely grade of differentiation, tumor necrosis, desmoplasia, lymphocytic infiltration and elastosis. The ER was detected in tissues using the avidin-biotin immunoperoxidase technique associated with the H222 monoclonal antibody from Abbott. All 39 biopsies were of infiltrating ductal carcinoma of breast and 16 of them expressed ER. The statistical analysis showed that the expression of ER was correlated with histological findings of good prognosis as well differentiated carcinomas, no tumor necrosis, absence or mild lymphocytic infiltration around the tumor cells and severe elastosis.

Breast↗

PubMed1991

Detection of human papillomaviruses in paraffin-embedded condylomata acuminata--comparison of immunohistochemistry, in situ hybridization, and polymerase chain reaction.

The polymerase chain reaction (PCR) is used for detection of human papillomavirus (HPV) DNA in paraffin-embedded tissue sections of condylomata acuminata. Incorporation of biotinylated nucleotides during the amplification process allows a highly sensitive, fast, and non-isotopic detection of viral DNA in a subsequent Southern dot blot. In 100% (13 of 13) of histologically confirmed condylomata, HPV-6 or -11 could be detected by polymerase chain reaction. By in situ hybridization 77% (10 of 13) and by immunohistochemistry (IHC) 69% (nine of 13) positive results were obtained. Because HPV genital infection is linked to penile and cervical dysplasia, polymerase chain reaction provides a powerful and highly sensitive tool for epidemiologic studies on sexual transmission of HPV.

Adult↗

PubMed2001

Diagnosis of aneuploidy in archival, paraffin-embedded pregnancy-loss tissues by comparative genomic hybridization.

OBJECTIVE: To evaluate the detection of aneuploidy in archival tissues from miscarriages by a method that uses microdissection and DNA extraction of villus cells from paraffin blocks, followed by universal DNA amplification and comparative genomic hybridization (CGH). DESIGN: Retrospective analysis. SETTING: Academic medical center. PATIENT(S): Nine archival tissues from cases of spontaneous abortion with trisomy 16 (two cases), trisomy 21 (three cases), trisomy 22 (two cases), triploidy (one case), and monosomy X (one case). INTERVENTION(S): Villus DNA was extracted from microdissected, formalin-fixed, paraffin-embedded tissues. Aneuploidy was detected by CGH after universal amplification of the DNA with the use of degenerate oligonucleotide-primed polymerase chain reaction. MAIN OUTCOME MEASURE(S): Detection of aneuploidy in archival pregnancy-loss tissues using CGH. RESULT(S): In all nine cases, DNA was successfully extracted from the microdissected tissues and was of sufficient quantity and quality for evaluation by CGH. In six of nine cases, the chromosomal abnormality detected by conventional cytogenetic analysis was identified by CGH: trisomy 16 (2/2), trisomy 21 (3/3), and trisomy 22 (1/2). One case of each of the following was not detectable: triploidy (1/1), monosomy X (1/1), and trisomy 22 (1/2). CONCLUSION(S): We propose CGH as a method for determination of aneuploidy in pregnancy-loss archival tissues when conventional cytogenetic analysis is unsuccessful or when it was not performed when fresh tissue was available.

Abortion, Spontaneous↗

PubMed1985

Immunohistochemical studies on human brain tumors using anti-Leu 7 monoclonal antibody in paraffin-embedded specimens.

Using the four-step peroxidase-antiperoxidase (PAP) method, the presence of the antigen recognized with anti-Leu 7 monoclonal antibody was investigated in paraffin-embedded human brain tissue and tumors. The antigen was demonstrated in the myelin sheaths, oligodendrocytes, and some choroid plexus cells in normal brain and in oligodendrogliomas, some astrocytomas and choroid plexus papillomas. The technique can be used to identify normal and neoplastic oligodendrocytes.

Antibodies, Monoclonal↗

PubMed1991

Flow-cytometric analysis of the DNA-content in paraffin-embedded tissue from colorectal carcinomas and its prognostic significance.

The nuclear DNA content of 163 colorectal carcinomas was determined by flow-cytometry (FCM) on formalin-fixed, paraffin-embedded tissue. DNA-aneuploidy was found in 97 cases (59.5%), in which no statistically significant correlations with sex, mean age, tumour stage (Dukes and pTNM) and tumour grade were noted. The frequency of aneuploidy was significantly higher in patients less than 70 years of age (p less than 0.01) and in tumours localized in the left colon and rectum (p less than 0.002), irrespective of their stage. The tumours in which different areas could be analysed (n = 80) showed a heterogeneous DNA-ploidy pattern in 18%. Comparison of the DNA content in primary tumours and in lymph node metastases (n = 49) showed a difference in DNA-ploidy in 38% of the DNA-aneuploid tumours, but in only 6% of the DNA-diploid carcinomas (p less than 0.02). DNA-aneuploid carcinomas tended to show a higher rate of local recurrence and were associated with an unfavourable prognosis (p = 0.04) in those patients in which complete resection of their tumours was possible (n = 72). The significantly higher mortality of patients with DNA-aneuploid carcinomas of stage pT3, as well as those with Dukes stage A and B tumours indicates that DNA-aneuploidy may be a stage-independent additional risk factor in colorectal cancer.

Aged↗

PubMed2000

Immunohistochemical staining of dystrophin on formalin-fixed paraffin-embedded sections in Duchenne/Becker muscular dystrophy and manifesting carriers of Duchenne muscular dystrophy.

We succeeded in immunostaining of monoclonal anti-dystrophin antibodies on formalin-fixed and paraffin-embedded muscle sections from patients with Duchenne muscular dystrophy, patients with Becker muscular dystrophy, and manifesting carriers of Duchenne muscular dystrophy using Catalyzed Signal Amplification(TM) system. The Catalyzed Signal Amplification system is an extremely sensitive immunohistochemistry staining procedure based on the peroxidase-catalyzed deposition of a biotinylated phenolic compound. We used three mouse monoclonal antibodies: DYS1, DYS2, and DYS3. Muscle sections were treated using the Target Retrieval Solution(TM) and the Catalyzed Signal Amplification system. In control patients, DYS1 and DYS2 were stained at the sarcolemma, but DYS3 remained unstained. In Duchenne muscular dystrophy patients, DYS1 and DYS2 staining were undetected. In Becker muscular dystrophy patients, the immunolabeling of DYSI and DYS2 were weak and discontinuous. In manifesting carriers of Duchenne muscular dystrophy, DYS1 and DYS2 staining showed a mosaic pattern of dystrophin-positive fibers and dystrophin-negative fibers. DYS1 and DYS2 staining patterns of this study are similar to those of frozen sections using conventional methods previously reported. In cases from whom frozen muscle sections cannot be obtained, immunohistochemical dystrophin analysis using the Catalyzed Signal Amplification system will be beneficial for the diagnosis and the screening of neuromuscular diseases.

3,3'-Diaminobenzidine↗

PubMed1980

Demonstration of species difference of placental alkaline phosphatase isozymes in acetone-fixed paraffin-embedded tissues.

The best preservation and discrimination of alkaline phosphatase isozymes by means of amino acid inhibition and heat treatment was obtained in sections of acetone-fixed paraffin-embedded tissues as compared to tissues fixed either in 95% ethanol or standard formalin. The results are illustrated in a study differentiating the isozymes of human and mouse placenta.

Acetone↗

PubMed1994

[Comparison of histology, immunohistochemistry, RT-PCR, in situ hybridization, and in situ RT-PCR for demonstration of hepatitis C virus in paraffin-embedded liver biopsies].

To compare immunohistochemical and molecular methods for the detection of hepatitis C virus (HCV) infection in archival liver biopsies we analyzed formalin-fixed and paraffin-embedded liver specimens of 10 patients with serologically confirmed HCV infection. Methods employed included indirect FITC-immunofluorescence, reverse-transcriptase polymerase chain reaction (RT-PCR) using extracted RNA and Southern blotting with chemiluminescence-based detection, non-radioactive in situ hybridization (ISH) with digoxigenin-labeled oligo- and cRNA probes, direct in situ RT-PCR with incorporation of labeled nucleotides into PCR-products, and indirect in situ RT-PCR using subsequent ISH for the visualization of intracellular PCR-products. Our results indicate that: (1) using the histological criteria described by Lefkowitch et al. (Gastroenterology 1993; 104-595) together with clinical data, most chronic HCV infections can be diagnosed by conventional histology, if liver biopsies are representative; (2) the commercially available mAB TORDJI-22 appears to cross-react with non-HCV epitopes; (3) molecular methods performed on routinely fixed and processed liver biopsies frequently yield false negative results due to sampling problems, low viral copy number and RNA degradation in infected cells; (4) analysis of HCV-RNA by RT-PCR of extracted total RNA is more sensitive than indirect in situ RT-PCR or ISH; and (5) direct in situ RT-PCR is not reliable despite the use of modifications such as DNase pretreatment and hot-start procedures. Further studies are required to define both optimal methods for sample processing and improvements of protocols, in order to increase detection sensitivity and specificity of HCV infection by immunohistochemical and molecular methods.

Biopsy↗

PubMed1991

Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.

Antibodies, Monoclonal↗

PubMed1978

Cryofracture of paraffin-embedded heart muscle cells.

A comparative study of internal cellular structures of the sheep ventricular myocardium has been conducted by scanning electron microscopy (SEM) and by transmission electron microscopy (TEM). Access to the cell interior for three-dimensional studies was obtained by cryofracturing paraffin-embedded tissue frozen in liquid nitrogen. For accurate localization of structures of special interest thick paraffin sections were examined in the light microscope (LM). Based on the information gained, it was possible to fracture the block in a desired plane. The fracturing was carried out by a light blow to a precooled scalpel held against the surface of the block, which was immersed in liquid nitrogen. After thawing and deparaffinizing at room temperature in several baths of xylene, the tissue pieces were critical point dried using CO2. As xylene was found to be miscible with CO2, it also served as an intermediate fluid. This method resulted in good preservation fo the myofibrils, mitochondria, sarcoplasmic reticulum and transverse tubules (T-tubules), which was confirmed by TEM studies of conventionally prepared tissue and of tissue originally prepared for SEM.

Animals↗

PubMed1991

Detection of enteroviral infection in paraffin-embedded tissue by the RNA polymerase chain reaction technique.

A method using the RNA polymerase chain reaction technique to diagnose infection retrospectively using single-stranded RNA enteroviruses in paraffin-embedded tissue blocks is reported. This method takes advantage of extreme sequence conservation in the 5' untranslated region of the enteroviral genome and uses two rounds of amplification with nested oligonucleotide primers, thus allowing rapid diagnosis without the use of radioactive reagents. The technique should prove useful in cases in which viral infection is suspected after histopathologic evaluation, when fresh or frozen tissues often are unavailable. The sensitivity of the method is demonstrated by successful amplification of Enterovirus 11 RNA extracted from 4-year-old liver tissue obtained at autopsy examination that was initially fixed and embedded 45 hours after death.

Base Sequence↗

PubMed1994

Detection of human papillomavirus type 16 DNA sequences in paraffin-embedded tissues from the female urinary tract.

We investigated the presence of human papillomavirus-related DNA sequences (HPV 6, 11, 16 and 18) in 33 formalin-fixed paraffin-embedded biopsies from the urinary tract of female patients with recurrent and persistent urethritis and cystitis, using the polymerase chain reaction (PCR). The samples for PCR reaction were selected among tissues examined for histological diagnosis on the basis of the presence of microscopic changes consistent with HPV infection. Sequences homologous to HPV 6, 11 and 18 genome were not found, while HPV 16-related DNA sequences were identified in 25/33 lesions with histopathological diagnosis of metaplasia (1 from the urethra, 23 from the trigone and 1 from the bladder). The results suggest that the spread of HPV in the female urinary tract may not be uncommon and point to the need for further research on the possible pathogenic role in recurrent female disturbances.

Cystitis↗