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Demonstration of hepatitis B-surface antigen in liver biopsies. A comparative investigation of immunoperoxidase and orcein staining on identical sections of formalin fixed, paraffin embedded tissue.

Staining of 1000 consecutive liver biopsies with orcein showed positive reaction in the cytoplasm of ground-glass hepatocytes in 18 of the biopsies. On new sections of the 18 orcein positive biopsies immunoperoxidase staining was performed in order to demonstrate hepatitis B-surface antigen (HBsAg). After destaining the same sections were stained with orcein. All biopsies showing positive orcein staining showed positive immunoperoxidase reaction. The number of positive cells was in biopsies with less than 20 positive cells per mm2 biopsy larger using the immumoperoxidase staining than with orcein staining. Further the staining contrast was more pronounced. Immunoperoxidase staining thus seems more sensitive than orcein staining for demonstrating HBsAg in liver tissues. Orcein stains HBsAg in liver tissue even though the antigen determinants are blocked by antibodies.

Cytoplasm↗

[Immunophenotyping of B lymphoma in bone marrow biopsies. Contribution of 4 monoclonal antibodies used on paraffin-embedded tissues:DDB.42,DNA.7,DNA.44 and DND.53].

An immunohistochemical study was performed on 132 routinely processed trephine biopsies of the bone marrow (40 reactive lymphocytosis, 80 B-cell lymphomas, 7 T-cell lymphomas, 5 Hodgkin's diseases), using 4 monoclonal antibodies, DBB.42, DNA.7, DBA.44, DND.53, which are directed against B-lymphocyte associated antigens, not destroyed by fixation. DBB.42, DNA.7 and DBA.44 are formalin and Bouin resistant. They do not stain myeloid, erythroblastic, histiomonocytic and endothelial cells, and are particularly suitable for bone marrow biopsies. DBB.42 and DNA.7 in association, identify all B-cell lymphomas and no T-cell lymphoma. DBB.42 recognizes respectively 65/65 and 13/15 low and high grade malignancy B-cell lymphomas, and DNA.7, 54/65 and 15/15. In our hands, these 2 antibodies are more reliable than L26 and MB2 on bone marrow biopsies. DBA.44 identifies 15/15 hairy-cell leukemias, and interestingly stains only 10% of other B-cell lymphomas, and no T-cell lymphoma. DND.53 is less specific, but reveals Reed-Sternberg cells. Combined immunostaining with anti T-lymphocyte antibody CD3, let us appreciate the contribution and the limits of immunohistochemistry in the diagnosis of bone marrow lymphoproliferative diseases, especially for interstitial and nodular lymphoid infiltrates of undetermined significance. These results point out the value of these 4 monoclonal antibodies in routinely processed bone marrow biopsies, particularly of DBB.42 and DBA.44.

Antibodies, Monoclonal↗

A new polychrome stain and simultaneous methods of histological, histochemical and immunohistochemical stainings performed on semithin sections of Bioacryl-embedded human tissues.

We describe a new polychrome stain and simultaneous methods of histological, histochemical and immunocytochemical staining performed on sections from human tissues embedded in the new hydrophilic resin Bioacryl. The polychrome stain involves the sequential use of Harris' Haematoxylin, silver methenamine, Light Green and Eosin or Safranin dyes and provides a highly specific visualization of the overall cytological tissue architecture. When histochemical, immunocytochemical, and polychrome stains are performed together on the same section, crisp images are obtained, yielding simultaneous data of histochemical and immunological reactivities with clear tissue architecture.

Acrylic Resins↗

Enzyme histochemistry on normal and pathologic paraffin-embedded lymphoid tissues.

So far, enzyme histochemical examination has been applied, with few exceptions, either to tissue imprints or to cryostat sections of freshly collected samples. This procedure is not easily applicable in routine histopathologic examination. In this study, a simplified tissue embedding procedure is presented which can be performed using an automatic tissue changer. The paraffin embedded samples can be used for both conventional histopathologic examination and for demonstrating enzymes in sections. The enzymes studied (alkaline phosphatase, alpha-naphthyl acetate esterase, acid phosphatase, tartrate resistant acid phosphatase, ATPase, peroxidase, and chloroacetate esterase) gave comparable results in formalin-fixed cryostat sections and paraffin sections in both normal and pathologic lymphoid samples. The only exception was ATPase, which could not be demonstrated on paraffin-embedded material. The technic described has broad application in the analysis of lymphoid diseases.

Acid Phosphatase↗

Lack of asbestos contamination of paraffin.

OBJECTIVE: To investigate possible asbestos contamination of paraffin and migration by asbestos fibers during the tissue-embedding process. DESIGN: Three sample categories were included in the study: (1) commercially available paraffin samples; (2) procedural control samples, which were prepared by processing the paraffin through the use of standard solvents and instruments; and (3) samples taken from areas adjacent to embedded tissue and evaluated for migration of asbestos from the tissue into the surrounding paraffin. The analysis of collected material from all samples was performed with analytical transmission electron microscopy. RESULTS: Only one extremely small tremolite fiber was found in any of the commercially available samples of paraffin. No asbestos fibers were found either in the procedural control samples or in the samples taken adjacent to the embedded lung tissue. CONCLUSIONS: First, it was extremely unlikely that any of the commercial paraffin samples would have skewed data due to embedded tissue. Second, the processing and instrumentation was not found to contribute asbestos material to the paraffin during the preparations. Finally, embedded tissue that contained high numbers of fibers, both uncoated fibers and asbestos bodies, did not contribute asbestos to the adjacent paraffin.

Asbestos↗

Quantitative comparison of pretreatment regimens used to sensitize in situ hybridization using oligonucleotide probes on paraffin-embedded brain tissue.

Paraffin embedding of tissue is generally perceived to dramatically reduce RNA detectability. As a consequence, in situ hybridization on paraffin-embedded tissue is largely confined to detection of high-copy RNA species (e.g., viral RNA) and/or to detection using typically more sensitive cDNA probes or riboprobes. In this study, several procedures for in situ hybridization on paraffin-embedded rat tissue using oligonucleotide probes complementary to cellular transcripts were developed and quantitatively compared. Certain pretreatments showed marked increases in sensitivity compared to untreated sections. Furthermore, through quantitative assessment using image analysis, sensitivity of optimal pretreatments was equal to that of routinely used fresh-frozen, postfixed tissue sections. The development of such techniques permitting in situ hybridization to be carried out on paraffin-embedded tissue allows a comparison of protein and mRNA distribution to be made in adjacent sections and provides the potential for double labeling by in situ hybridization and immunohistochemistry which may not be possible on post-fixed frozen sections.

Animals↗

Immunohistochemical detection of cytokines in paraffin-embedded mouse tissues.

We have successfully developed a method for the immunohistochemical detection of interleukin 2 (IL-2), IL-4, IL-6, IL-10O, IFNgamma and TNFalpha using monoclonal antibodies (MAb), in sections of mouse tissue embedded in paraffin wax. The method involved fixation in periodate-lysine-paraformaldehyde (PLP), rapid dehydration and infiltration under vacuum with paraffin wax at 54 degrees C. Comparative observations demonstrated that the method gives equivalent or better results than formaldehyde fixed, frozen sections. Since reliable controls, both positive and negative, are paramount for interpretation of immunohistochemical staining, such controls were determined. The following tissues were shown to be suitable as positive controls when using paraffin-embedding: spleen for the detection of TNFalpha, small intestine for IL-2, IL-4 and IL-10, and HSV-1 infected eyes for IL-6 and IFNgamma. We conclude that PLP fixation and low temperature paraffin-embedding is a method which provides both preservation of excellent tissue morphology and reliable immunohistochemical identification of cytokines. These attributes will be invaluable in a wide variety of experimental situations.

Animals↗

Glycol methacrylate in light microscopy: nucleic acid cytochemistry.

Techniques utilizing Feulgen, azure B bromide, methyl green-pyronin, gallocyanin chromalum and cresyl violet stains have been modified and adapted for visualizing nucleic acids in 0.5-2.0 micrometer sections of tissues embedded in glycol methacrylate (GMA). Methods for evaluating the stain specificity for DNA and RNA using deoxyribonuclease and ribonuclease digestions, aldehyde blocking, and acid extractions are also described. The specificity of the stains in GMA embedded tissues is comparable to that reported for paraffin-embedded tissues.

Acrylates↗

A methenamine-silver staining technique for glomerular basement membranes in plastic-embedded renal tissue.

A method is described for the staining of glomerular basement membranes of aldehyde-fixed, osmicated renal tissue embedded in Epon or Araldite. The procedure presented does not require chemical digestion of the polymerized Epon or Araldite matrix before application of the methenamine-silver technique. There is no divergence from the procedures for fixation and embedding for transmission electron microscopy (TEM). This enables sections from the same block of renal tissue to be examined both by light microscopy (LM) and TEM without loss of time or sacrifice of quality.

Animals↗

Codon 178 mutation of the human prion protein gene in a German family (Backer family): sequencing data from 72-year-old celloidin-embedded brain tissue.

Familial Creutzfeldt-Jakob disease was first described in a family from northern Germany in the 1920s (Backer family). PCR amplification of DNA extracted from brain tissue embedded in celloidin 72 years ago shows a GAC to AAC substitution at codon 178 of the prion protein gene. This mutation is associated with fatal familial insomnia and familial Creutzfeldt-Jakob disease in a number of families of diverse ethnic background.

Aged↗

The use of osmicated tissues for Lowicryl K4M embedding.

Tissue chopper slices of rat incisor, rat parotid gland, and chicken tibiae, fixed with 1% glutaraldehyde, were post-fixed with potassium ferrocyanide-reduced osmium tetroxide, dehydrated with methanol, and conventionally embedded in Lowicryl K4M at -20 degrees C. The tissues showed an ultrastructural appearance comparable with that of their Epon-embedded counterparts and, in particular, the Golgi apparatus was well defined. Furthermore, Lowicryl K4M-embedded osmicated tissues permitted both post-embedding lectin-gold cytochemistry and immunogold labeling.

Acrylic Resins↗

Attempts to quantitate immunocytochemistry at the electron microscope level.

The ability to localize intracellular macromolecules in situ by high resolution techniques has been made possible by the development of antibody labelling of thin sections obtained either from tissues embedded in an hydrophilic matrix, or by ultracryotomy or from conventional plastic embedded tissue. When particle-tagged immunological reagents are used to visualize intracellular antigens, quantitative information can be obtained by combining particle counts with morphometric estimations of compartment volume. Various detection systems have been used successfully for quantitation, which include ferritin-conjugated antibodies, biotin-avidin-ferritin complexes and, more recently, gold-protein A conjugates. Examples of the use of these techniques the localization of secretory proteins in pancreatic exocrine cells, opsin and a large membrane protein in photoreceptor cells of frog retina, and contractile proteins in skeletal muscle are given. Quantitative data obtained by morphometric analysis, both in bovine and rat pancreatic exocrine cells, are compared with values assessed by biochemical methods.

Amylases↗