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At least 1,063 records · Page 59Linked to original sources
PubMed1997

Immunohistochemical detection of Haemophilus parasuis serovar 5 in formalin-fixed, paraffin-embedded tissues of experimentally infected swine.

An avidin-biotin complex immunohistochemistry technique was developed to detect Haemophilus parasuis serovar 5 in experimentally infected 18-21-day-old conventional pigs, using a rabbit polyclonal antiserum. Seven of 10 intratracheally inoculated animals developed a low to medium degree of fibrinous polyserositis; meninges and pleura were the most severely affected areas. Haemophilus parasuis was recovered from 9 of 10 pigs; in 2 of them H. parasuis was isolated from tracheal swabs only. Positive immunohistochemistry results, mainly observed as free bacteria or bacteria within inflammatory cell cytoplasm in the fibrinopurulent exudate, were observed in 8 of 10 animals. Cross-reactivity with Actinobacillus pleuropneumoniae was detected but not with other gram-positive and gram-negative bacteria tested. This immunohistochemistry technique seemed to be at least as sensitive as microbiologic cultures and could be useful in studies of pathogenesis and retrospective diagnosis. However, cross-reactivity with A. pleuropneumoniae means that positive immunohistochemistry results in lung tissue from field cases would be dubious.

Animals↗
PubMed1981

Prolonged incubation time in immunohistochemistry: effects on fluorescence staining of immunoglobulins and epithelial components in ethanol- and formaldehyde-fixed paraffin-embedded tissues.

By prolonging the incubation time from 30 min to 20 hr at room temperature, fluorochrome conjugates may be applied at about ten times higher dilution and yet produce specific immunofluorescence staining of enhanced intensity. This modification of the direct method is important for reagent economy and, in addition, affords improved staining features for all antigens tested in formaldehyde-fixed tissues. It is particularly valuable when paired staining is used to characterize lymphoproliferative B-cell processes in pathological routine material; a clear-cut distinction between polyclonal and monoclonal expression of cytoplasmic immunoglobulin (Ig) is obtained, and cells giving rise to a false staining pattern are easily pinpointed. It is likewise advantageous to use prolonged incubation with conjugate for the localization of Ig-producing and Ig-bearing B cells in saline-extracted ethanol-fixed tissues, and the same holds true for Ig and C3 in immune-complex deposits. Also IgE on the surface of mast cells in tissues from atopic subjects is visualized distinctly with this modification. However, the localization or epithelial components is not consistently improved in ethanol-fixed tissues when the incubation time is prolonged; secretory products such as lactoferrin, lysozyme, amylase, and secretory component (SC) are not always immobilized sufficiently by ethanol fixation to avoid diffusion artifacts and a substantial loss from the cytoplasm. Differences in intracellular storage probably contribute to the variable antigen stability.

Epithelium↗
PubMed1981

Immunocytochemical detection of peptides in osmicated and plastic-embedded tissue. An electron microscopic study.

In an attempt to improve the ultrastructural preservation of tissue immunostained with the postembedding staining technique, we verified the effect of postfixation with OsO4 on the antigenicity of several pituitary hormones and neuropeptides. For this purpose, the rat pituitary and central nervous system (CNS) were perfused with 2.5% glutaraldehyde and postfixed in two different ways: a) postfixation by immersion of small fragments in a mixture of 1% OsO4 and 1% potassium ferrocyanide, and b) postfixation with perfusion of 500 ml of 0.5% OsO4. The results obtained were similar with the two types of postfixation. In the pituitary gland, all the hormones could be very easily detected, although the staining was less intense for the glycoprotidic hormones. In the CNS the following neuropeptides: somatostatin, luteinizing hormone-releasing hormone (LHRH), Leu-enkephalin and substance P could be immunostained. These results indicate that a variety of polypeptidic antigens, including small neuropeptides, can survive postfixation with OsO4. In both the pituitary and CNS, the ultrastructural preservation was very good with a high contrast of membranes, thus permitting a clear identification of positive organelles. Moreover, it appears that, since the synaptic junctions are well-preserved after postfixation with OsO4, postembedding staining can now be utilized to identify and classify the different categories of endings containing neuropeptides.

Animals↗
PubMed1982

Double labeled-antigen method for demonstration of intracellular antigens in paraffin-embedded tissues.

A double "labeled-antigen" method has been developed for the simultaneous staining of both kappa and lambda light chains in fixed paraffin sections. The method is a two step procedure utilizing a mixture of antisera against kappa and lambda light chains in the first stage, followed by the addition of a mixture of kappa antigen labeled with horseradish peroxidase and lambda antigen labeled with alkaline phosphatase. The selection of substrates yielding reaction products of contrasting color enabled the observer to distinguish kappa-containing cells (brown) from lambda-containing cells (blue). Reactive plasma cells stained either pure brown (kappa) or clear blue (lambda) in a ratio of 1.5:1. Blood vessels containing serum immunoglobulins showed a mixed brown-blue reaction, as did the Reed-Sternberg cells of some cases of Hodgkin's disease. The advantages of this double labeled-antigen method over previously reported methods for achieving double staining are discussed.

Antigens↗
PubMed1982

Immunohistochemical identification of mast cells in paraffin- and epon-embedded tissues using platelet factor 41.

An immunoperoxidase method has been developed for staining heparin in the granules of mast cells. The method employs human platelet factor 4 (or anti-heparin) and a rabbit antiserum to this polypeptide. Platelet factor 4 binds to mast cell heparin and provides the basis for immunoperoxidase staining using the rabbit antiserum. Preliminary studies of mast cells in various tissues indicate that the stain is quite specific for the content of mast cell granules, presumably heparin and possibly other glycosaminoglycans.

Blood Coagulation Factors↗
PubMed1983

Immunolabeling of carbonic anhydrase isoenzyme C and glial fibrillary acidic protein in paraffin-embedded tissue sections of human brain and retina.

The specificities of carbonic anhydrase isoenzyme C (CA C) and glial fibrillary acidic (GFA) protein as immunocytochemical markers for different glial cell populations in human brain and retina were studied using indirect immunofluorescence and peroxidase-antiperoxidase complex methods. With antibodies against CA C, only those cerebral cells that were morphologically oligodendrocytes and Müller cells of the retina showed positive immunostaining reaction, whereas antibodies against GFA protein selectively labeled cerebral astrocytes and a part of the glial cells and fibers in the inner layers of the retina. In double labeling, when both glial cell markers were successively localized in the same cerebral tissue sections, GFA protein immunofluorescence was never found in the immunoperoxidase-stained CA C-positive cells, which further supports the oligodendrocyte-specificity of CA C in human brain.

Brain↗
PubMed1986

A single-step silver enhancement method permitting rapid diagnosis of cytomegalovirus infection in formalin-fixed, paraffin-embedded tissue sections by in situ hybridization and immunoperoxidase detection.

A single-step silver enhancement method was developed which intensifies the polymerized nickel-complexed diaminobenzidine (Ni-DAB) reaction product of peroxidase. With such enhancement, an in situ hybridization procedure can be performed in less than 8 hr by using a 2-hr hybridization incubation and direct detection. Cytomegalovirus (CMV)-infected lung sections were hybridized in situ for 2 hr with a biotinylated CMV genomic-length probe. The probe was detected directly with avidin-biotinylated peroxidase using Ni-DAB as the substrate, and the reaction product was enhanced with silver. Silver was deposited only on the Ni-DAB and not on normally argyrophilic substances. Indirect detection of the probe using a sandwich technique before silver enhancement proved more sensitive, but the length of the procedure was increased without substantially changing the result (infection vs. no infection). Sensitivity was also improved by omitting the dehydration step before applying the probe, and by increasing the temperature and duration of denaturation. In a blinded study of 21 open-lung biopsies, 13 of 13 culture-negative specimens were negative by hybridization, and 7 of 8 culture-positive specimens were positive by hybridization. Modified short hybridization with a biotinylated probe and silver-enhanced direct detection therefore provides a rapid but sensitive method for diagnosis of viral infection.

3,3'-Diaminobenzidine↗
PubMed1988

Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue.

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.

Animals↗
PubMed1989

Localization of alpha 2u-globulin within protein droplets of male rat kidney: immunohistochemistry using perfusion-fixed, GMA-embedded tissue sections.

We investigated the light microscopic subcellular localization and quantitation of alpha 2u-globulin (alpha 2uG) in male rat kidney after 2,2,4-trimethylpentane exposure using a monoclonal antibody to alpha 2uG. Slices of perfusion-fixed kidney were cold-processed in glycolmethacrylate and the antigen localized after an avidin-biotin-horseradish peroxidase procedure with Hanker-Yates reagent as the chromagen. Light microscopic examination revealed resolution comparable to low-magnification electron microscopy, with excellent morphological detail of tissue architecture, including subcellular localization of alpha 2uG within lysosomes of P2 segment cells of the proximal tubule epithelium. The anatomical relationship between alpha 2uG and TMP-induced protein droplets observed after staining with Lee's methylene blue-basic fuchsin was studied using serial sections. Image analysis of selected P2 segments in treated and control rats revealed a high correlation between subcellular localization of alpha 2uG and protein droplet deposition in the cytoplasm of P2 segment cells of the proximal tubule epithelium. Quantitative morphometry of alpha 2uG-stained proximal tubule epithelium 72 hr after treatment with 50 mg/kg 2,2,4-trimethylpentane p.o. demonstrated a 1.5- to 2-fold increase in staining area of tubules from treated rats compared with controls. Similar increases of Lee's methylene blue-basic fuchsin-stained protein droplets were also observed, but quantitative morphometry of the protein droplets was technically more difficult owing to a lower staining contrast between droplets and the surrounding cytoplasm. This immunohistochemical procedure provides a valuable technique for further studies on the pathological role of alpha 2uG in protein droplet nephropathy of male rats induced by many environmental chemicals, and demonstrates the value of cold glycolmethacrylate processing to improve morphological detail.

Alpha-Globulins↗
PubMed1995

Detection of P53 expression and S-phase cell fraction in paraffin-embedded tissue by a double-labeling technique.

TP53 is a gene that normally regulates cell growth and division. Alterations to it may induce a proliferative advantage and confer an aggressive phenotype. In breast cancer, we observed a poor correlation (rs = 0.17) between P53 expression and proliferative activity evaluated as [3H]-thymidine ([3H]-dT) labeling index and an independent prognostic relevance of the two variables. We used a double-labeling technique to simultaneously evaluate the fraction of P53-positive and [3H]-dT-labeled cells to analyze the degree of association between the two markers on individual cells in order to understand their biological significance. The study was performed on a series of 44 P53-positive (P53+) breast cancers. Histological sections were immunostained for P53 with monoclonal antibody (MAb) PAb1801 and then processed for autoradiography. A weak direct relation between P53 positivity and [3H]-dT incorporation (rs = 0.4) was observed on the overall series of P53+ tumors and was maintained in subgroups defined by several biological and pathological features, except for estrogen receptor-negative tumors. The simultaneous presence of P53 expression and [3H]-dT incorporation was directly and significantly proportional to the fraction of S-phase cells of the tumor (rs = 0.7). Conversely, the fraction of cells expressing only P53 was inversely related to cell proliferation (rs = -0.66). These findings support the hypothesis that P53 has biological functions other than cell cycle regulation.

Antibodies, Monoclonal↗