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PubMed1987

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application.

This paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryostat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution. Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal anti-type IV collage antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections. It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.

Animals↗
PubMed2000

No evidence of somatic activating mutations on gonadotropin receptor genes in sex cord stromal tumors.

OBJECTIVE: To search for somatic activating mutations of gonadotropin receptor (FSH-R and LH/chorionic gonadotropin receptor [CG-R]) genes as a cause of sex cord stromal tumors. DESIGN: Molecular studies in human tissue. SETTING: University hospital. SPECIMEN(S): Eight granulosa cell tumors collected from paraffin-embedded tissue, eight Leydig cell tumors, and three thecomas collected from fresh-frozen or paraffin-embedded tissue. INTERVENTION(S): Tumor samples were used for DNA extraction. The entire exon 11 of the LH/CG-R gene and a hot spot for gonadotropin receptor activating mutations on exon 10 of the FSH-R gene were amplified by polymerase chain reaction. The former was analyzed by denaturing gradient gel electrophoresis and automatic direct sequencing, and the latter by automatic direct sequencing. MAIN OUTCOME MEASURE(S): Results of denaturing gradient gel electrophoresis and automatic direct sequencing. RESULT(S): No somatic activating mutation was detected in exon 11 of the LH/CG-R gene in eight Leydig cell tumors and three thecomas. In addition, no mutations were detected in eight granulosa cell tumors in the hot spot for activating mutations in exon 10 of the FSH-R gene. CONCLUSION(S): Somatic activating mutations of gonadotropin receptors seem to play no relevant role in the development of sex cord stromal tumors.

Exons↗
PubMed1980

Evaluation of staining methods of resin embedded cutaneous tissue sections of mycosis fungoides.

Biopsies from lesional skin of patients with histologically confirmed mycosis fungoides were embedded in resin and semi-thin ( I micrometer) sections were submitted to a variety of staining procedures, including Toluidine blue, Richardson's Huber, Giemsa, Paragon and Tribasic stains. The stained sections were examined with the light microscope and the quality of cellular morphological detail afforded by the different stains was assessed. The Toluidine blue, Richardson's and Paragon stains were found to be technically easy to perform and gave the best representation of nuclear and cytoplasmic detail Of these, the authors favoured Richardson's stain. The technique of processing the tissue, and staining a mounting procedures are detailed.

Adolescent↗
PubMed1993

Image analysis versus flow cytometry for DNA ploidy quantitation of solid tumors: a comparison of six methods of sample preparation.

With the availability of user-friendly interactive image analysis instruments for DNA analysis, there is a growing need for comparison with the established methodology of flow cytometry. We have compared the results of DNA ploidy quantitation in 12 solid tumors prepared by six different techniques of sample preparation: flow cytometry of fresh cell suspensions and of nuclei isolated from formalin-fixed, paraffin-embedded tissue; and image analysis of touch preparations, of disaggregated cells from paraffin-embedded tissue as well as of 3- and 7-microns-thick tissue sections. Complete agreement in DNA ploidy results obtained by the six methods was found in six out of 12 solid tumors. Image analysis of touch preparations detected most tetraploid and multiple aneuploid peaks. Sections of 7-microns-thick tissue gave better histogram quality than 3-microns-thick sections, however tetraploid peaks were not resolved in one case. Image analysis of disaggregated paraffin-embedded tumor showed comparable ploidy to fresh touch preparations in seven out of 12 cases, the discrepancies being due to loss of tetraploid or multiple aneuploid peaks. Flow cytometry gave good histograms, but tetraploid and multiple aneuploid peaks were occasionally not detected. Each method presents advantages and disadvantages. Flow cytometry and image analysis are complementary methods for DNA quantitation, and more than one method may be necessary to confirm the DNA content of solid tumors.

Cell Nucleus↗
PubMed2004

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer.

BACKGROUND: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. METHODS: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. RESULTS: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. CONCLUSIONS: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.

Animals↗
PubMed1995

Differential expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human breast and skin tissue: in situ mapping of cell type-specific expression.

O6-Alkylguanine is a major toxic, mutagenic, and carcinogenic lesion in cellular DNA that is repaired by O6-alkylguanine-DNA alkyltransferase (ATase). The expression of this gene is directly related to the cellular sensitivity of alkylating agents, with levels of this protein varying widely among human organs, tumors, and cell types. To better understand specific cell-type responses to repairing O6-alkylguanine lesions in DNA, we used colorimetric in situ hybridization, with an ATase-specific antisense oligomer probe, to map the cellular distribution of ATase mRNA in tissue sections of normal adult human breast and neonatal foreskin tissues. This is the first report of mapping ATase gene expression directly in normal human breast and skin tissues. Paraffin-embedded tissue sections were hybridized with a digoxigenin-labeled, 39-base antisense ATase oligomer. Hybridization of the probe to cells expressing the ATase gene was visualized after immunodetection with an alkaline phosphatase-conjugated anti-digoxigenin antibody. After color development, we simultaneously identified tissue architecture and cell types and measured the expression of the ATase gene. There was no hybridization-specific color when sections were mock hybridized, hybridized with a sense probe, or treated with RNase. In the breast tissue, 93% of the cells in the loosely connective tissue and 84% of the myoepithelial cells expressed high levels of ATase mRNA. Most of the luminal ductal epithelial cells (61%) were devoid of stain, indicating undetectable levels of ATase mRNA. In skin dermis, 93% of the fibroblasts appeared to express high levels of ATase mRNA. Within the epidermis, approximately 64% of the basal and 65% of the granular epithelial cells expressed ATase mRNA. Expression was undetectable in the epithelial cells of the suprabasal layer of the epidermis. There was very little interindividual variation (< 17%) in the distribution of expression of ATase within the same cell types of different individuals. These data illustrate the differential potential of individual cell types within the organ matrix to repair O6-alkylguanine damage in cellular DNA. This data may provide insights into the understanding of cell type-specific responses to carcinogens.

Breast↗
PubMed2003

Applications of Fourier transform infrared microspectroscopy in studies of benign prostate and prostate cancer. A pilot study.

Fourier transform infrared (FTIR) microspectroscopy has been applied to a study of prostate cancer cell lines derived from different metastatic sites and to tissue from benign prostate and Gleason-graded malignant prostate tissue. Paraffin-embedded tissue samples were analysed by FTIR, after mounting onto a BaF(2) plate and subsequent removal of wax using Citroclear followed by acetone. Cell lines were analysed as aliquots of cell suspension held between two BaF(2) plates. It was found that the ratio of peak areas at 1030 and 1080 cm(-1), corresponding to the glycogen and phosphate vibrations respectively, suggests a potential method for the differentiation of benign from malignant cells. The use of this ratio in association with FTIR spectral imaging provides a basis for estimating areas of malignant tissue within defined regions of a specimen. Initial chemometric treatment of FTIR spectra, using the linear discriminant algorithm, demonstrates a promising method for the classification of benign and malignant tissue and the separation of Gleason-graded CaP spectra. Using the principle component analysis, this study has achieved for the first time the separation of FTIR spectra of prostate cancer cell lines derived from different metastatic sites.

Adenocarcinoma↗
PubMed1995

Demonstration of Epstein-Barr virus genomes, using polymerase chain reaction in situ hybridization in paraffin-embedded lymphoid tissues.

We used the polymerase chain reaction (PCR) in situ hybridization (ISH) (PCR-ISH) on sections of malignant lymphoma and nonspecific lymphadenitis to detect small amounts of Epstein-Barr virus (EBV), a DNA virus of the herpes virus family. We first surveyed the EBV DNA by Southern blot analysis and PCR, and then compared results of the two PCR/ISH methodologies with the results of simplified/sensitive ISH for the positive cases. The target of the simplified in situ (DNA-ISH) was a few copies of EBV DNA per cell, and the target of the sensitive in situ (RNA-ISH) was as many as 10(7) copies of EBV RNA per cell. When EBV DNA was detected by Southern blot, DNA-ISH, RNA-ISH and PCR-ISH all revealed EBV genomes. When PCR revealed only amplified EBV DNA, DNA-ISH showed no EBV genomes, but PCR-ISH and RNA-ISH showed EBV genomes in a few cells. When PCR showed no detectable amplified EBV DNA, all of DNA-ISH, RNA-ISH and PCR-ISH showed no genomes. These findings indicate that PCR-ISH consistently detected a few copies of the EBV virions. The PCR-ISH was as sensitive as RNA-ISH. The RNA-ISH could not detect virus if RNA was not expressed, but the PCR-ISH could detect virus without such expression. The ability to detect a single copy of a specific gene in situ has many advantages and multiple applications in molecular biology, pathology, and cell biology.

Base Sequence↗
PubMed1983

Diagnosis of human lymphoma with monoclonal antileukocyte antibodies.

Two monoclonal antibodies have been produced that react with antigens present on human white cells. These reagents differ from other monoclonal antibodies of similar specificity in that the antigens they recognize are resistant to conventional tissue-fixation and embedding procedures. These reagents can therefore be used in immunocytochemical staining of paraffin-embedded tissue sections. We assessed the practical usefulness of this technique in the histopathological diagnosis of human lymphoid neoplasms by staining a wide range of routine surgical biopsy specimens of normal and neoplastic tissue (gathered from five institutions), using an indirect immunoperoxidase technique. In all 40 cases of non-Hodgkin's lymphoma, positive labeling of neoplastic cells was obtained with one or both antibodies. In contrast, no staining of neoplastic cells was observed in 60 samples of nonlymphoid neoplasms. We conclude that many of the difficulties encountered by histopathologists in distinguishing between lymphoid and nonlymphoid neoplasms may be overcome by immunohistologic labeling with monoclonal antibodies such as the ones we have studied.

Animals↗
PubMed1991

Spontaneous ameloblastic fibro-odontoma in a female mouse.

A spontaneous ameloblastic fibro-odontoma is described in a 73-wk-old female mouse. The tumor was located in the molar mandibular region. The diagnosis was made on the basis of the histologic presence of odontogenic epithelial tissue embedded in mesodermal tissue resembling dental papilla, and containing in addition dentin and enamel. The positive reaction for keratin and vimentin confirmed the presence of ectodermal and mesodermal elements respectively in the tumor.

Animals↗
PubMed1985

Light and electron microscope localization of G-17- and G-34-like immunoreactivities of human gastrinomas.

Gastrin- 17 (G-17) and gastrin-34-like immunoreactivities of human gastrinoma cells were investigated at light and electron microscope level using N-terminally directed antisera. The procedure includes (a) the 24 hr/immunoperoxidase staining of Bouin-fixed paraffin embedded tissues, (b) the immunoelectron microscopic labelling of aldehyde-fixed Epon-embedded tissues according to the immunogold technique. On light microscopy, a variable number of tumor cells stained for G-34. In contrast, G-17 immunoreactivity was very low or undetectable in the tumor material, although it was easily detected in endocrine cells of similarly processed human pyloric mucosa. On electron microscopy, most of the tumor cell granules belonging to the round compact or dense-cored type exhibited a variable labelling for G-34, whereas the vacuolar/floccular type remained unlabelled. In contrast, the labelling for G-17 occurred over most of the tumor cell granules, whether compact or floccular. Dense granules of varying size and shape, previously shown to store C-terminal gastrin immunoreactivity, were only faintly labelled by the two antisera. When compared to the labelling pattern of human pyloric G-cells, the predominance of round dense granules with G-34 and G-17 immunoreactivity in gastrinoma cells suggests an incomplete or defective post-translational processing of the precursor peptide.

Colloids↗
PubMed1979

Freezing of tissue-limits for the autoradiographic localization of diffusible substances.

Frozen thin sections and sections from freeze-dried and embedded tissue are used for the autoradiographic localization of diffusible substances at the electron microscope level. The presence of ice crystals in such sections may limit the autoradiographic resolution. Ice crystals are formed during freezing and may grow during subsequent processing of tissue. The contribution of ice crystal growth to the final image was estimated by measuring the distribution of the ice crystal sizes in freeze-etch replicas and in sections from freeze-dried and embedded tissues. A surface layer (10-15 mu) without visible ice crystals was present in both preparations. Beneath this surface layer the diameter of ice crystals increased towards the interior with the same relationship between crystal size and distance from the surface in the freeze-etch preparation as in the freeze-dry preparation. Ice crystal growth occurring during a much longer time during freeze-drying compared to freeze-etching does not significantly contribute to the final image in the electron microscope. The formation of ice crystals during freezing determines to a large extent the image (and therefore the autoradiographic resolution) of freeze-dry preparations and this probably holds also for thin cryosections of which examples are given.

Animals↗
PubMed1978

Histochemical identification of T and B areas in paraffin-embedded lymphoid tissues by demonstration of alpha-naphthyl acetate esterase (ANAE) activity.

A strong activity of acid alpha-naphthyl acetate esterase (ANAE) in T lymphocytes and macrophages within mouse lymphoid organs is demonstrable even after paraffin-embedding, if a rapid embedding procedure with acetone as dehydrating agent is used. The lymphoid tissue structure is better preserved in paraffin sections procured after this rapid embedding procedure than in frozen sections commonly used for histochemical demonstration of ANAE. In the paraffin sections the pattern of the ANAE reaction allows a clear delineation of the T and B areas in the lymphoid organs, and the intracellular location of the focal ANAE activity in T lymphocytes is readily discernible.

Animals↗
PubMed1998

Fluorescence in situ hybridization shows spatial distribution of as yet uncultured treponemes in biopsies from digital dermatitis lesions.

Fluorescence in situ hybridization (FISH) was performed on sections of plastic-embedded tissue using 16S rRNA-directed oligonucleotide probes to visualize uncultured treponemes in skin biopsies of cows with digital dermatitis. Plastic as embedding material allowed sectioning of hard and soft tissue with a defined thickness, avoiding the risk of dragging bacteria into the tissue while sectioning. furthermore, it provided a good signal-to-noise ratio. Using this method the spatial distribution of three different bacterial phylotypes was visualized simultaneously within the tissue. Whereas debris covering the ulcers contained a mixture of different micro-organisms, a layering of certain treponemal phylotypes was observed deeper in the epidermis. Confocal laser scanning microscopy and subsequent three-dimensional reconstruction of series of optical sections confirmed that the treponemes migrated intercellularly around the cells, most of them directed towards the dermis. In situ hybridization on tissue embedded in plastic proved to be a useful method to study mixed bacterial infections since it combines excellent histological conservation of tissue with identification of bacterial species by simultaneous use of probes labelled with different fluorescent dyes. This technique may have implications for in situ detection, identification and localization of microorganisms in veterinary as well as in human medicine.

Animals↗
PubMed2003

Detection and identification of fungi from fungus balls of the maxillary sinus by molecular techniques.

The aim of this study was to find a reliable method for the detection and identification of fungi in fungus balls of the maxillary sinus and to evaluate the spectrum of fungi in these samples. One hundred twelve samples were obtained from patients with histologically proven fungal infections; 81 samples were paraffin-embedded tissue sections of the maxillary sinus. In 31 cases, sinus contents without paraffin embedding were sent for investigation. PCR amplification with universal fungal primers for 28S ribosomal DNA and amplicon identification by hybridization with species-specific probes for Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus glaucus, Pseudallescheria boydii, Candida albicans, and Candida glabrata were performed for all samples. Furthermore, PCR products were sequenced. Fresh samples were also cultivated. Fungal DNA was detected in all of the fresh samples but only in 71 paraffin-embedded tissue samples (87.7%). Sequence analysis was the most sensitive technique, as results could be obtained for 28 (90.3%) fresh samples by this method in comparison to 24 (77.4%) samples by hybridization and 16 (51.6%) samples by culture. However, sequence analysis delivered a result for only 36 (50.7%) of the paraffin-embedded specimens. Hybridization showed reliable results for A. fumigatus, which proved to be the most common agent in fungus balls of the maxillary sinus. Other Aspergillus species and other genera were rarely found.

DNA, Fungal↗
PubMed1983

The influence of embedding media and fixation on the post-embedment ultrastructural demonstration of complex carbohydrates. I. Morphology and periodic acid-thiocarbohydrazide-silver proteinate staining of vicinal diols.

The influence of fixation and embedding medium on the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining reactivity in the mouse intestine was studied. It was found that the combination of osmium tetroxide and epoxy resins was the least sensitive for the demonstration of complex carbohydrate with the PA-TCH-SP method. Post-osmication reduced, but did not abolish, PA-TCH-SP reactivity (except for the Golgi complex) when non-epoxy resins were used. The staining pattern of a particular organelle differed depending on the embedding medium used. Golgi cisternae exhibited the most intense PA-TCH-SP reactivity in non-osmicated tissues embedded in non-epoxy resins. Post-osmication of tissues was required to reveal the fine structure of the glycocalyx as well as to preserve the fine structure of tissues embedded in styrene-methacrylate and styrene-Rigolac 2004. The choice of fixation procedures and embedding media in a given study should be governed primarily by the sites of interest.

Animals↗
PubMed1993

Interphase cytogenetics in paraffin sections of lung tumors by non-isotopic in situ hybridization. Mapping genotype/phenotype heterogeneity.

To better understand the relationship between specific chromosome changes found in human lung tumors and their phenotypic consequences at the tissue level, an in situ hybridization procedure was optimized for use on formalin-fixed paraffin-embedded tissue sections of human lung tumors. Pretreatment heating of sections, pepsin concentration, duration of pepsin treatment, hybridization conditions, and posthybridization washing conditions were varied to determine optimum conditions. The deparaffinized sections were stained with centromeric probes for chromosomes 7 and 17, and a chromosome index for each tumor was derived by dividing the mean number of chromosome signals found on the tumor cells by the mean number of chromosome signals on normal cells (lymphocytes and fibroblasts) in the same section. This chromosome index was then compared with the DNA index determined in an adjacent section by Feulgen staining followed by image analysis quantitation. The chromosome index correlated well with the DNA index, but in some cases, chromosome 7, or 17 in other cases, was either over- or under-represented compared with the corresponding DNA index. In addition, chromosome and DNA alterations were shown to be differentially expressed within the same tissue section, correlating with a change in tumor differentiation status. These results suggest that in situ hybridization will prove to be an important tool for determining the underlying genetic basis for tissue phenotype heterogeneity by allowing genetic determinations to be made on paraffin-embedded tissue sections where tumor histological architecture is preserved.

Carcinoma, Non-Small-Cell Lung↗