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PubMed1994

Preferential localization of c-kit product in tissue mast cells, basal cells of skin, epithelial cells of breast, small cell lung carcinoma and seminoma/dysgerminoma in human: immunohistochemical study on formalin-fixed, paraffin-embedded tissues.

Eighteen hundred and eighty-four cases of human solid tumours and 833 samples of normal human tissues, formalin-fixed and paraffin-embedded, were examined immunohistochemically for expression of c-kit oncogene product using polyclonal antibody against synthesized c-kit peptide. Seminoma/dysgerminoma and small cell lung carcinoma (SCLC) show preferential c-kit expression at 92% and 36% frequency, respectively, whereas only sporadic cases of cervical carcinoma and non-SCLC lung carcinoma show c-kit positivity. A normal tissue counterpart positive for c-kit product is detected in the testis (spermatocyte) and ovary (oocyte) but not in the lung or the cervix. In contrast, normal epithelial cells of the breast, skin basal cells and tissue mast cells harbour c-kit product, but transformed cells of the former two are largely deficient in the c-kit protein. One hundred and thirty-nine neuroendocrine tumours and 39 non-pulmonary small cell carcinomas were all negative, except for two cases of neuroblastoma. This indicates a distinct character for SCLC in c-kit expression. The c-kit product may be a useful marker in diagnostic pathology of seminoma/dysgerminoma and SCLC among human solid tumours, and in distinction of SCLC from non-pulmonary small cell carcinoma.

Adult↗
PubMedDate not supplied

Tissue subtraction DNA in situ hybridization (TSDISH): in situ detection of DNA abnormalities in formalin-fixed paraffin-embedded tissue sections by subtraction hybridization with whole genomic DNA.

We have developed a novel in situ histochemical method of screening for genetic alterations of human malignancies by subtraction hybridization of genomic DNA without employing specific probes to give a colorimetric reaction. We identified a t(13;14) chromosome abnormality in the chromosome spread of a patient with multiple myeloma. In situ hybridization of a whole cell preparation of MCF-7 cell demonstrated reaction products as intranuclear dots-in all MCF-7 cells. We subsequently examined the cells of different foci of esophageal squamous cell carcinoma(10) and esophageal biopsy specimens (9) by this method. Hybridization with genomic DNA from the patients demonstrated no reaction products in the stromal cells of the esophagus. However, hybridization with reference DNA from a healthy individual demonstrated intranuclear reaction products in the stromal cells, possibly due to individual genomic differences. There were more intranuclear reaction products in the carcinoma cells than in the stromal cells when hybridized with reference DNA. When hybridized with the reference DNA above, the cells of the non-pathologic epithelium of 8 of 10 malignant esophagi demonstrated significantly more reaction product than the stromal cells of the some specimens. This was not detected in the cells of normal epithelium obtained from non-cancerous esophagi suggesting the accumulation of genetic alterations of the non-malignant epithelium of the cancerous esophagus. This method is thought to detect DNA alterations, including those which have not been previously identified, using genomic DNA for hybridization, and the results can be correlated with the morphological findings. Application of this in situ method, together with other molecular genetic techniques may contribute to the analysis of various genetic alterations of human malignancies using archival material.

Chromosome Aberrations↗
PubMed1993

False aneuploidy in flow cytometric DNA analysis of paraffin embedded tissue: effects of Carnoy's fixation.

False aneuploidy was detected on flow cytometric DNA analysis of paraffin embedded axillary lymph nodes negative for tumor. It was hypothesized that "clearing" of axillary fat in Carnoy's solution to facilitate lymph node dissection might be responsible for false aneuploidy. Various tissues fixed overnight in Carnoy's were compared to formalin fixed paraffin embedded controls. Under these conditions no false aneuploid peaks were detected, but Carnoy's fixation did shift the G0/G1 histogram peak to the left, increase the G0/G1 CV and increase the S phase fraction relative to formalin fixed controls. It was then hypothesized that partial fixation of nodes in Carnoy's followed by formalin fixation might result in false aneuploid peaks. Twenty-two lymph nodes were partially fixed in Carnoy's for periods ranging from 5 to 60 min followed by complete fixation in formalin. Seven of these nodes did show false aneuploid peaks. By contrast, no aneuploidy was detected in formalin fixed controls. It was concluded that tissues in contact with Carnoy's solution may be a source of false aneuploidy and/or false elevation of S phase fraction. This reinforces the need for matched negative tissue controls for DNA analysis of paraffin embedded specimens whenever possible.

Acetates↗
PubMed1979

Immunofluorescent localization of pea storage proteins in glycol methacrylate embedded tissue.

Improved immunofluorescent techniques have been developed for the high resolution light microscopic localization of intracellular antigens in plant tissue. Thin sections of pea cotyledon tissue which had been fixed in paraformaldehyde and embedded in glycol methacrylate were reacted with mono-specific antibodies to the storage proteins legumin and vicilin. These antibodies were raised in sheep, purified by affinity chromatography and tested by immunoelectrophoresis and immunodiffusion. Using the indirect technique, rhodamine-labeled antibodies permitted specific fluorescent localization of the legumin and vicilin to small (ca. 1 micrometer) cytoplasmic organelles in near mature tissue. Subsequent histochemical staining verified the proteinaceous nature of these organelles. Parameters affecting staining specificity and background fluorescence are discussed.

Fluorescent Antibody Technique↗
PubMed1981

The demonstration, by immunofluorescence, of Legionella pneumophila organisms in human lung tissue embedded in epoxy resin.

Legionella pneumophila serogroup 1 has been demonstrated by indirect immunofluorescence in human lung tissue fixed in 10% formal saline and embedded in epoxy resin. The organism was visualised using Rabbit serogroup 1 antibody and Sheep anti rabbit fluorescein conjugate. The fluorescent labelled organism was found distributed throughout the tissue with focal areas in alveolar spaces and also visualised by electron microscopy in the same tissue. This method therefore enables specific identification of the L. pneumophila organism with the antiserum and also affords the opportunity of studying the bacterium ultrastructurally in the same tissue.

Epoxy Resins↗
PubMed1991

Effects of different fixatives on detection of nucleic acids from paraffin-embedded tissues by in situ hybridization using oligonucleotide probes.

Detection of nucleic acids from paraffin-embedded material by in situ hybridization with oligonucleotide probes is increasingly being used. To determine the effect of fixation on the preservation of DNA and mRNA, we studied 18 lymphoid tissues fixed in B5, formalin, OmniFix, ethanol, and Bouin's fixatives and embedded in paraffin by in situ hybridization, using biotinylated oligonucleotide poly d(T) probes and immunoglobulin light chain probes. Detection of DNA using the poly d(T) probe was most consistent and most intense in tissue fixed in formalin, followed by OmniFix and ethanol, with B5 and Bouin's fixatives yielding unsatisfactory results. Detection of mRNA, using the light chain probes, was most consistent and most intense with tissue fixed in formalin and Bouin's solution, followed by B5 fixative, with OmniFix and ethanol fixatives yielding unsatisfactory results. The results of mRNA detection using the poly d(T) probe were found not to correlate with mRNA content as determined by the light chain probes for several fixatives, possibly owing to selective degradation of portions of the mRNA molecule.

Acetates↗
PubMed1986

Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

Actin Cytoskeleton↗
PubMed1974

The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody.

A method is described for the demonstration of specific immunoglobulin in plasma cells and other lymphoid cells in sections taken from routine surgical histology specimens which have been formalin fixed and paraffin embedded. An indirect sandwich technique was employed using specific rabbit antihuman immunoglobulin antisera (anti-K, L, G, A, and M) and a swine antirabbit serum Ig G, conjugated with horseradish peroxidase. The presence of plasma cells was revealed by staining the tissue-bound peroxidase-labelled antibody, having previously stained the endogenous peroxidase a contrasting colour. It was possible to demonstrate clearly immunoglobulin in the plasma cells of tissues processed and embedded several years previously. Some of the potential uses of the method are discussed.

Animals↗
PubMed1997

Immunohistochemical detection of p21WAF1/CIP1 and p53 proteins in formalin-fixed paraffin-embedded tissue sections of squamous cell carcinoma of the skin.

Thirty-two cases of squamous cell carcinoma (SCC) of the skin were investigated as to the expression of p53 and p21 (WAF1/CIP1) using an immunohistochemical method. These cases were surgically resected or biopsied, tissue samples were then fixed in formalin and embedded in paraffin in the conventional way. Microwave heating was used for antigen retrieval. The primary monoclonal anti-p21 antibody and the monoclonal antibody against p53 were employed. The labeled-streptoavidin-biotin-peroxidase method was used for immunohistochemical staining. Of these, 30 cases showed overexpression of p53 staining, but normal epidermal cells were free of stain. p21 positive cells were detected faintly in the middle layer cells of normal epidermis. Of these, 30 cases showed overexpression of p21 staining. The staining pattern of p53 and p21 showed intratumoral heterogeneity in SCC. In general, there was the inverse relationship between p21 and p53 staining in tumors, namely p53 positive cells were p21 negative and vice versa. However, some of the tumor cells expressed both genes simultaneously. This study supports a hypothesis that p21 expression is regulated by p53, and that it is also regulated by an additional pathway(s) in SCC.

Antibodies, Monoclonal↗
PubMed1986

Binding specificity of two monoclonal antiglioma antibodies: immunocytochemical studies using a new tissue embedding technique.

The binding specificities of two monoclonal antiglioma antibodies (MAB) derived from hybrids GE2 and BF7 (Schnegg et al. 1981) were tested by indirect immunofluorescence (IF) and two immunoperoxidase (IP) methods. Studies were done on biopsies from 33 human CNS tumors, human derived glioma cells, and N-ethylnitrosourea-induced neurogenic rat cells in culture. The immunohistochemical reactions of MAB were investigated in snap-frozen tumor material, conventionally paraffin-embedded material, and tumors embedded in formol sucrose/gum sucrose/paraffin (FSGSP) by the new tissue processing technique of Bolton and Mesnard (1982), which preserves and enhances the antigenicity of tissues. The FSGSP processing offered a better immunocytochemical staining with MAB as compared to cryostat material, while the conventionally embedded paraffin sections of tumors did not stain at all. The binding of MAB revealed an affinity to both glial tumor cells and normal astrocytes. The techniques described are suitable for the identification of an astrocytic subpopulation within gliomas, and may improve the understaining of antigen expression in various stages of astrocytic dedifferentiation.

Animals↗
PubMed2000

T311--an anti-tyrosinase monoclonal antibody for the detection of melanocytic lesions in paraffin embedded tissues.

Tyrosinase is a key enzyme in melanin biosynthesis and represents a marker of melanocytic differentiation. We previously generated T311, a murine monoclonal antibody to the tyrosinase recombinant protein. This study was performed to evaluate T311 as a diagnostic immunohistochemical reagent for use on formalin-fixed paraffin-embedded pathological material. We analyzed the specificity of the antibody on a panel of normal and neoplastic tissues, and we assessed its sensitivity in a large number of metastatic and primary malignant melanomas, nevi, three angiomyolipomas, and two vitiligo specimens. T311 revealed intense reactivity on paraffin-embedded material. Immunoreactivity was limited to cells of melanocytic differentiation and no immunostaining was present in unrelated normal tissues and tumors. Eighty-four percent of metastatic malignant melanomas were immunoreactive with T311 and showed predominantly a homogeneous expression pattern. However, in primary melanomas of the desmoplastic/spindle cell type, T311 revealed a poor immunoreactivity. Nevi showed intense staining at the junctional zone, while the dermal component revealed decreasing reactivity towards deeper areas. Only one angiomyolipoma was focally immunoreactive with T311. Vitiligo specimens were immunonegative. We conclude that T311 is a specific and sensitive marker for the detection of melanocytic lesions in formalin-fixed paraffin-embedded tissues and a useful serological reagent for diagnostic pathology.

Antibodies, Monoclonal↗
PubMed1998

Detection of the SYT-SSX chimeric RNA of synovial sarcoma in paraffin-embedded tissue and its application in problematic cases.

We report the development of a reverse-transcriptase polymerase chain reaction assay that detects (in paraffin-embedded, formalin-fixed tissue) the SYT-SSXchimeric RNA transcript resulting from the t(X;18) of synovial sarcoma. The primers chosen detect both of the SSX1 and SSX2 partners, and the target sequence is small enough (87 base pairs) to be reliably detected in archival and variably processed consultation material. To demonstrate its usefulness, we applied it to 14 problematic cases, including spindle cell tumors of the thoracic region, of the neck, and of subcutaneous tissue. For instance, we show that, depending on the location, synovial sarcoma can mimic malignant solitary fibrous tumor, the spindle epithelial tumor with thymus-like differentiation, or skin adnexal tumors. Molecular detection of the SYT-SSX chimeric RNA should allow the reclassification of difficult cases in which the morphologic features overlap different entities or in which tumor nosology is still evolving.

Adult↗
PubMed1997

[Detecting pathogens of fetal defects in paraffin embedded tissues by polymerase chain reaction].

OBJECTIVE: To examine 3 types of pathogens in dead fetus with congenital defects for exploring the pathogenesis. METHODS: The paraffin embeded brain and liver tissues from 39 fetal autopsies with congenital defects were examined for toxoplasma, cytomegalovirus, and herpes simplex II by polymerase chain reaction. Among them 16 placentae were examined as well. RESULTS: Pathogens were detected in 25 of 39 cases, a positive rate of 64%. Nine out of the 13 fetuses with urogenital defects were positive for the shove pathogens, while 8 from the 12 with neural tube defects were positive as well. CONCLUSION: Intrauterine infection was one of the important causes of birth defects. The characteristics of placenta morphologic changes were described, and the significance of placenta investigation in congenital malformation was emphasized.

Animals↗
PubMed1991

Detection of a monocyte/macrophage differentiation antigen in routinely processed paraffin-embedded tissues by monoclonal antibody Ki-M1P.

A new monoclonal antibody Ki-M1P that is raised against supernatants of detergent solubilized human lymph node tissue is described. Ki-M1P recognizes in particular monocytes and their macrophage derivatives as tested by light- and electron-microscopic immunohistochemistry. Granulocytes, dendritic cells as the accessory cells of humoral and cellular immune response, and epithelial, endothelial, neural, and mesenchymal cells do not react with Ki-M1P. In extensive application Ki-M1P has proven to be a useful marker for distinguishing monocytic leukemias within FAB groups M4 and M5. The recognized antigen is composed of five proteins with molecular masses of about 60, 92, 98, 124, and 150 kDa in blood monocytes, whereas tissue macrophages tested so far expressed only the 60-kDa protein. Because the Ki-M1P antigen is not destroyed or masked during routine fixation and paraffin embedding of biopsy tissue samples, Ki-M1P represents a useful diagnostic reagent for the identification of physiological functional and pathologic reaction forms as well as neoplastic variants of the human monocyte/macrophage system even in retrospective studies.

Animals↗
PubMed1980

Glial fibrillary acidic protein in the retina of the developing albino rat: an immunoperoxidase study of paraffin-embedded tissue.

The peroxidase-anti-peroxidase method was used on paraffin-embedded material to demonstrate the distribution of glial fibrillary acidic (GFA) protein, an astrocyte-specific protein, in the developing retina of the albino rat. At birth activity was scant and was confined to scattered, poorly differentiated cells in the inner retinal layers near the optic disc. At 3 days primitive astrocytes which displayed GFA protein activity were confined to the stratum opticum near the optic disc. With increasing age these cells were found at greater distances from the optic disc and began to assume the appearance of typical fibrous astrocytes. By 30 days the perikarya of these cells were confined almost exclusively to the region between the nerve fiber layer and the inner limiting membrane. The processes of these cells terminated either in suckerlike end-feet upon blood vessels or, to a lesser extent, ended in relation to axon fascicles of the nerve fiber layer. A second population of GFA protein-active cells existed as perivascular glia which were found upon vessels in the inner portion of the stratum opticum in young animals. In the mature retina perivascular glia were found on vessels throughout the stratum opticum and in the inner portion of the inner plexiform layer. Unequivocal staining of Müller cells or their processes was not obtained. The best staining was obtained with fixatives containing minimal concentrations of aldehydes, especially in tissue from younger animals. The fixative which gave the best preservation of cellular structure along with preservation of GFA protein antigenicity was Perfix (Fischer Scientific Company).

Animals↗
PubMed1997

Interphase cytogenetic (in situ hybridization) analysis of astrocytomas using archival, formalin-fixed, paraffin-embedded tissue and nonfluorescent light microscopy.

Astrocytomas contain nonrandom chromosomal abnormalities that recently have been correlated with shortened patient survival. Two frequently reported aberrations are trisomy 7 and monosomy 10. We assessed the numerical complement of chromosomes 7 and 10 in formalin-fixed, paraffin-embedded brain biopsy tissue from 28 diffuse astrocytomas by in situ hybridization using a nonfluorescent enzymatic detection system. Clinical follow-up of at least 5 years was available in 26 cases (93%). Monosomy 10 was identified in 7 cases (25%): astrocytoma, 1 case; anaplastic astrocytoma, 1 case; and glioblastoma, 5 cases. Trisomy 7 was identified in 11 cases (39%): astrocytoma, 5 cases; glioblastoma, 6 cases. Multivariate analysis revealed that monosomy 10 was the most statistically significant negative predictor of patient survival. Numerical chromosomal abnormalities are detectable in astrocytomas in archival tissue using interphase cytogenetics and nonfluorescent light microscopy. Although larger studies are required, our data suggest that potentially useful prognostic information may be obtained with this approach.

Adult↗
PubMed1982

Modifications of embedding tissues in durcupan ACM (Fluka) based on viscosity index measurements.

Viscosity of Durcupan ACM mixtures without as well as with accelerator was measured, at temperatures of 25, 40, and 50 degrees C respectively. The time course of polymerization was expressed within first few hours following admixture of accelerator, at temperature mentioned above. On the basis of our findings a modified embedding schedule for tissues (peripheral autonomic nerves and ganglia) has been recommended: infiltration in Durupan 1 (without accelerator) could be performed at 50 degrees C, when viscosity of embedding mixture is greatly reduced, to 190 mPa. s. Further infiltration in Durcupan 2 (with accelerator added) is carrier out at 40 degrees C; the initial viscosity of embedding medium at this temperature is 440 mPa. s. and even after 4 hours it keeps below 200 mPa. s. So the infiltration time could be prolonged in contrast to that at 50 degrees C, which usually brings better embedded blocks and better ultrathin sections.

Animals↗
PubMed1981

Specific identification of hemoglobin and myoglobin in renal tubular casts by the fluorescent antibody technic on fixed embedded tissues.

Hemoglobin and myoglobin have been identified in formalin-fixed paraffin-embedded sections of human autopsy material with the indirect fluorescent antibody technic. The sensitivity and specificity observed were comparable to those reported by other investigators who used fresh-frozen sections. When this approach was applied to archival autopsy tissue, it was possible to identify some pigmented renal tubular casts specifically as myoglobin or hemoglobin and to separate them from other pigmented casts such as bile or melanin. The data suggested that the hemeproteins, as they pass along the nephron, are progressively denatured, and that their reactivities with specific antisera, iron stains, and peroxidase substrates are altered. Intact hemeprotein molecules react with specific antibody and show peroxidase activity, but the iron is not demonstrable by traditional methods. In the proximal tubules, after filtration, they react with specific antibody, show peroxidase activity, and also have demonstrable iron. By the time they reach the collecting tubules, they show only peroxidase activity.

Female↗