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At least 1,513 records · Page 84Linked to original sources
PubMed2003

Application of automated mRNA in situ hybridization for formalin-fixed, paraffin-embedded mouse skin sections: effects of heat and enzyme pretreatment on mRNA signal detection.

Recently, an automated mRNA in situ hybridization application was introduced for the Ventana Discovery instrument. The application was designed so that all necessary steps from baking through signal detection were completed within 1 day on the instrument. We applied this technology for visualizing the expression site of versican in formalin-fixed mouse skin paraffin tissue sections. Our focus of this study was to demonstrate the effects of protease digestion or heating pretreatment, termed cell conditioning, on the hybridization signal using a well characterized versican antisense riboprobe. Paraffin sections were automatically deparaffinized, fixed, and acid-treated. Then, the tissue sections were subjected to protease digestion alone (3 strengths), cell conditioning alone, or the combination of cell conditioning and protease digestion. Hybridization was performed with digoxigenin-labeled versican antisense probe (20 ng/slide) for 6 hours, and the signal was detected using a Nitro blue Tetrazolium chloride 5-Bromo-4-cloro-3-indolyl phosphate toluidine salt (NBT/BCLIP) substrate solution for 3 hours on the instrument. Cell conditioning alone did not produce any signal, whereas the highest strength of protease digestion produced noticeable background staining. However, when cell conditioning and mild protease digestion were combined, the signal for versican mRNA was clearly demonstrated in the hair papilla region. Thus, we demonstrated the effects of the cell conditioning step followed by mild protease digestion for enhancing the mRNA target staining compared with protease digestion or the cell conditioning step alone. We verified that the automated in situ hybridization process was applicable for formalin-fixed mouse skin paraffin sections and that the automated 1-day protocol is simple and reproducible. The precise control of automation allows fine tuning of temperature and enzyme dose to find the optimized assay condition for the signal to noise ratio and morphology.

Animals↗
PubMed1993

Assessment of c-erbB-2 amplification by immunohistochemistry in paraffin-embedded breast cancer.

The c-erbB-2 (HER-2/neu) proto-oncogenes is important in oncogenesis and for determination of prognosis in a number of human malignancies. DNA (Southern) hybridization and immunoblotting (Western) techniques are most commonly utilized for determining amplification status and protein expression of this proto-oncogene, respectively. These extraction techniques are often time-consuming, costly, and subject to variability depending on the histological characteristics of the tumor. Paraffin-immunohistochemistry (P-IHC), on the other hand, is time and cost-effective. In addition, this technique may offer enhanced sensitivity and specificity over extraction techniques due to the in situ nature of analysis. In data presented here, 67 cases of human mammary carcinoma were concomitantly assessed for c-erbB-2 gene copy number and oncoprotein expression by dilutional DNA hybridization and P-IHC, respectively. In 64 (95.5%) of 67 cases, high level expression was associated with gene amplification, whereas no detectable expression was associated with a normal diploid gene copy number. In two of the three discrepant cases, P-IHC predicted amplification not corroborated by Southern analysis. In these cases, tumor mass was limited by the intraductal component of the lesion or by an abundance of stromal elements within the specimen. We conclude that P-IHC offers a favorable alternative to Southern analysis in the assessment of c-erbB-2 gene copy number of this oncoprotein in human mammary carcinoma. Furthermore, immunohistochemistry may prove superior to either extraction technique in specimens with limited tumor mass, such as biopsy materials, stroma-rich tumors, or early stage lesions such as intraductal carcinoma.

Breast Neoplasms↗
PubMed2001

Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM).

Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.

Ammonia↗
PubMed1998

Genetic analysis of multiple loci in microsamples of fixed paraffin-embedded tissue.

Molecular analysis of alterations in genomic DNA is essential for understanding mechanisms by which chemical agents induce or modify tumor development. The assessment of microsatellite polymorphisms, loss of heterozygosity, mutations, and gene rearrangement allows specific comparisons of tumors to premalignant lesions or normal tissue or between similar tumors seen in laboratory species and humans. Utilization of these techniques is frequently limited by minute quantities of available tissue, often restricted to small formalin-fixed tumors or biopsies in paraffin blocks. To address these limitations, we have combined recently developed methodologies for selective recovery, amplification, and analysis of DNA. These techniques provide sufficient materials of high quality for analysis of DNA alterations in microscale amounts of starting material. By combining whole genome amplification through primer extension preamplification with locus-specific heminested PCR, we are able to analyze multiple genetic loci from as little as 1 mm2 of a 3-micron-thick formalin-fixed paraffin section. From 10 to greater than 100 loci can be analyzed per tissue section, and locus-specific PCR products may be further evaluated by a variety of techniques (e.g., SSCP, sequencing). Integrating these methodologies into situations where evaluation of very small tissue samples is necessary provides a powerful approach for elucidating molecular events that may be causally related to chemically induced cellular transformation and tumorigenesis.

Animals↗
PubMed1989

Morphometrical analysis of mycosis fungoides on paraffin-embedded sections.

Morphometry was carried out on H&E stained paraffin sections of 29 cases of contact dermatitis (CD) and 35 cases of mycosis fungoides (MF) (patch stage 12; plaque stage 11; tumor stage 12); 9 nuclear parameters, mean thickness of the infiltrate and 5 stereological parameters were assessed for each slide. Application of a non-parametric discriminant analysis (k-nearest neighbour method) which is based on median of nuclear areas, mean maximal nuclear diameter, volume density and numerical density of nuclei provided discrimination between CD and patch stage MF at an efficiency of 82.9% (specifity 86.7%, sensitivity 61.5%). Efficiency of discrimination between CD and plaque stage was 92.5% (specifity 89.7%, sensitivity 91.7%) and between CD and tumor stage 100% when discriminant analysis was based on the mean thickness of the infiltrate. Although unequivocal discrimination between CD and MF cannot be achieved in each individual case, morphometry on routine paraffin material obviously provides additional objective criteria for the diagnosis of early MF.

Biopsy↗
PubMed2006

Quantitation of chromosome 1p and 19q deletions in glial tumours by interphase FISH on formalin-fixed paraffin-embedded tissue.

Deletions on chromosomes 1p and 19q identify oligodendroglial tumours that are likely to have a complete response to some chemotherapy regimens and are associated with prolonged patient survival. Detection of these chromosomal alterations is becoming increasingly important in the evaluation of glial tumours. Interphase fluorescence in situ hybridisation (FISH) is one of a number of techniques for detecting deletions, and is an efficient method for screening large numbers of tumours. We used FISH to detect 1p and 19q deletions in formalin-fixed paraffin sections of 11 oligodendrogliomas, 11 oligoastrocytomas, two astrocytomas and four glioblastomas multiforme. 1p and 19q deletion and non-deletion ratios were tabulated in 200 nuclei in each tumour. We found considerable variation between tumours in the fraction of cells with deletions. This variation has not been reported previously and its clinical significance will be clarified with patient follow-up. There was little variation between regions within the same tumour.

Brain Neoplasms↗
PubMed2005

Inter-laboratory validation of PCR-based detection of Mycobacterium tuberculosis in formalin-fixed, paraffin-embedded tissues.

The present study is based on the initiative for quality assurance in pathology of the German Society of Pathology and the Professional Association of German Pathologists. Four panel laboratories with experience and expertise in polymerase chain reaction (PCR) detection of Mycobacterium tuberculosis were selected to establish the prerequisites for continuous external laboratory trials, in particular, by providing pre-tested specimens and evaluation criteria for participating institutes. In the first step, the four panel laboratories performed an internal trial to test their own reliability and reproducibility. Paraffin sections and DNA preparations from 34 tissues (25 clinical specimens and 9 controls) totalling to 66 samples were evaluated by each panel institute according to their own protocols. The methodologies differed and are described in detail. Despite these differences, a high degree of inter-laboratory reliability was achieved. In this report, we summarise our results including the correlation with the histology and provide recommendations for applying PCR-based methodology for the detection of mycobacterial DNA in surgical specimens. Supplementary data are available online at http://www.charite.de/ch/patho (rubric "Forschung"). Pre-tested specimens are now available for the external trial and can be ordered from the steering institute via Oligene (http://www.oligene.com/). All molecular pathology laboratories are invited to participate in this quality assurance initiative.

Animals↗
PubMed1995

Microwave irradiation of paraffin-embedded tissue sensitizes the TUNEL method for in situ detection of apoptotic cells.

Apoptosis is a morphologically distinct form of programmed cell death that plays an important role in the growth regulation of a variety of tissues and also in the elimination of self-reacting immunocompetent cells. Several techniques for the qualitative and quantitative detection of this process have been established; recently, an in situ nick end-labelling technique based on the detection of DNA fragmentation, which is a molecular characteristic of apoptotic cell death, was described. Applying this method to paraffin sections of human tissues, sensitivity was observed to be inconsistently low with regard to the expected number of apoptotic cells. In the present study we show that irradiation of the tissue sections in 10 mM citrate buffer, pH 6.0, by microwaves at 750 W considerably enhances the sensitivity of this nick end-labelling technique.

Apoptosis↗
PubMed1998

Immunocytochemical detection of somatostatin receptors sst1, sst2A, sst2B, and sst3 in paraffin-embedded breast cancer tissue using subtype-specific antibodies.

The long-acting somatostatin analogue octreotide (SMS 201-995) inhibits growth of certain breast cancer cell lines in vivo and in vitro. Because the antiproliferative action of octreotide depends on at least the presence of somatostatin receptors, it is crucial to determine the pattern of somatostatin receptor protein expression on the tumor cells. In the present study, we have raised polyclonal antibodies to somatostatin receptor subtypes (ssts) sst1, sst2A, sst2B, and sst3 using peptides corresponding to their COOH-terminal sequences. These antisera were used for immunocytochemical staining of paraffin sections of 33 primary breast cancers. Somatostatin receptor-like immunoreactivity (Li) was predominantly localized to the plasma membrane of the tumor cells. In the vast majority of positively stained tumors, somatostatin receptor-Li was uniformly present on nearly all tumor cells. Both the level and the pattern of expression of ssts varied greatly between individual carcinomas. sst2A-Li and/or sst2B-Li was detectable in 28 tumors (85%); among these, 14 tumors (42%) showed particularly high levels of sst2-Li. sst1-Li was found in 17 (52%) cases and sst3-Li in 16 (48%) cases. The expression of ssts was independent of patient age, menopausal status, diagnosis, histological grade, and levels of estrogen and progesterone receptors. The immunocytochemical determination of somatostatin receptor status allows direct detection of receptor protein on the tumor cells and, hence, may provide more precise information than reverse transcription-PCR for predicting response to octreotide therapy in breast cancer.

Adult↗
PubMed1997

Mel-5: a novel antibody for differential diagnosis of epidermal pigmented lesions of the skin in paraffin-embedded sections.

Histological evaluation of epidermal melanocytes on routine staining is difficult and cannot be made with accuracy. Widely known antibodies such as S-100 and HMB-45 are unreliable for normal epidermal melanocytes. Furthermore, S-100 stains other cells including Langerhans' cells. Results of incubation with DOPA are inconsistent and the procedure is time-consuming. We have evaluated the use of Mel-5, an antibody that was developed against melanoma and melanocytes. This antibody is a mouse monoclonal antibody that specifically detects a 75 kDa glycoprotein usually expressed by normal melanocytes, naevi and melanoma cells in routinely fixed paraffin sections. Histological differentiation between pigmented actinic keratosis in photodamaged skin and lentigo maligna can be difficult. The atypical keratinocytes, particularly in the basal layer, can be confused with atypical melanocytes, especially if they are pigmented. Similarly, distinctions between lichen planus-like keratosis and lichenoid melanoma in situ and lentigo maligna and lentigo may be difficult. Use of Mel-5 in such cases has shown consistent results in separating melanocytic from non-melanocytic lesions. This antibody is also helpful in evaluating biopsies of patients with vitiligo, post-inflammatory pigmentary alteration and regressed or regressing melanocytic lesions. Furthermore, Mel-5 is an invaluable tool in quantification of epidermal melanocytes in research projects.

Animals↗
PubMed2003

Estimation of hormone receptor status in fine-needle aspirates and paraffin-embedded sections from breast cancer using the novel rabbit monoclonal antibodies SP1 and SP2.

We describe a method of immunocytochemical assessment of estrogen receptor (ER) status on alcohol-fixed smears obtained by fine-needle aspiration (FNA) from breast cancer patients, using a commercially available rabbit monoclonal antibody anti-ER (SP1) without any antigen retrieval. A series of 40 aspirates were analyzed and the results of ER status were compared with the respective formalin-fixed tissue using the same procedure and with assessment by the classical method using the mouse monoclonal antibody 6F11 (anti-ER) with antigen retrieval on paraffin sections. Twenty-four out of the 40 cases examined were positive at least by two methods and 16 were negative for all three determinations. The results obtained in the ER immunocytochemical assay on aspirates and paraffin sections using the antibody SP1 and those obtained on paraffin sections using the antibody 6F11 were quite similar. In one case the material was insufficient to interpret the reaction in the cytological specimen and only one case, with focal positivity reaction on paraffin sections, was negative in the cytological specimen. The intensity of nuclei staining in cytological smears of breast cancer cells was stronger than that observed by traditional methods. We also assessed progesterone receptor (PR) status on 40 paraffin-sections from breast cancer patients, using a commercially available rabbit monoclonal antibody anti-PR (SP2), with the same characteristics described for anti-ER (SP1). The results were compared with assessment by the classic method with mouse monoclonal antibody 1A6 (PR) on paraffin sections and total agreement was observed. Of the 40 cases examined, 18 were positive and 22 were negative for the two determinations. We conclude that the application of the ER method on alcohol-fixed smears/paraffin sections with the rabbit monoclonal antibody SP1, and the PR method on paraffin sections with the rabbit monoclonal antibody SP2, provide several advantages, such as high sensitivity and specificity of the reaction, stronger immunostaining, shorter procedures times, and avoidance of antigenic retrieval methods.

Adenocarcinoma↗
PubMed1993

Detection of human papilloma virus (HPV) infection in paraffin-embedded tissues of endometrial carcinoma.

Although human papilloma virus (HPV) associated lesions constitute a well recognized clinical entity in the female lower genital tract, namely vulva, vagina and cervix, few studies have demonstrated HPV infection in other genital sites, particularly in the ovary and uterine corpus. Recently, with the highly sensitive polymerase chain reaction (PCR) technique, HPV infections were found in an ovarian tumour and adenocarcinoma of the cervix. This prompted a retrospective analysis of HPV DNA in 22 cases of endometrial adenocarcinoma in order to investigate the possible carcinogenesis of HPV in the uterine corpus. In this study DNA extraction was performed from paraffinized cancerous tissues and the normal cervical counterpart. HPV 6, 11, 16 and 18 primers specific oligonucleotides were used in PCR to detect the presence of this oncogenic virus. HPV 16 DNA was found in 1 endometrial adenocarcinoma and 4 cervical tissues. Our result did not support the aetiological role of HPV in the carcinogenesis of endometrial carcinoma.

Adenocarcinoma↗