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At least 19 recordsLinked to original sources
PubMed1990

Recovery of nuclei from glycol-methacrylate-embedded tissue.

The analysis of antigens, enzyme histochemical markers, and DNA has become an important part of the classification of some leukemias, lymphomas, and other neoplastic diseases. Many of the relevant antigens and most of the relevant enzyme histochemical activities are destroyed and others are less than optimally preserved in tissues embedded in hot paraffin. Most enzymatic activities and antigens are well preserved in tissues embedded at 4 degrees C in glycol methacrylate (GMA). The measurement of DNA content in neoplastic cells with the most commonly employed techniques depended on the availability of fresh suspensions of cells until the development by Hedley of methods that permit the recovery of nuclei from paraffin blocks for this purpose. In order to facilitate the analysis of antigens, enzymatic markers, and DNA from the same sample of tissue, we have developed a means of recovery of nuclei from GMA-embedded tissues. Twenty-microns-thick sections of GMA-embedded tonsil were either pretreated with an organic solvent (absolute ethanol or 2-ethoxyethanol) followed by rehydration in phosphate buffered saline (PBS) or directly rehydrated in PBS. The suspensions were formed mechanically by gentle sonication. The type of fixative and length of PBS rehydration were varied. Tissue fixed in 100% acetone, embedded in GMA, and rehydrated directly in PBS for six days gave the highest average yield of nuclei, 3.7 x 10(7) nuclei per gram tissue. In order to assess DNA binding of fluorescent dyes, 2-microns-thick GMA sections were stained with chromomycin, Hoechst 33342 (Sigma Chemical, St Louis, MO), and propidium iodide. Hoechst 33342 bound specifically to the nuclei with low background staining.

Benzimidazoles↗
PubMed1980

Stevenel's Blue, an excellent stain for optical microscopical study of plastic embedded tissues.

Stevenel's Blue is a reliable, rapid, and clean, one-step polychromatic stain for 1 micron thick epoxy sections. The staining solution, originally used by L. Stevenel (1918) to stain human parasites, is made by adding diluted potassium permanganate (2%) to an aqueous solution of the methylene blue (1.3%) and redissolving the precipitate thoroughly, by boiling in water bath and filtering. Staining is carried out in a Coplin jar at 60 degrees C for approximately 10 minutes for tissues embedded in Epon 812 or Poly 812, or 20 minutes for tissues embedded in Spurr's medium. The sections are rinsed, air dried, and mouted in Permount. The staining solution is very stable, and does not tend to form precipitates on the tissue. The stain brings excellent histological differentiation to nuclear, cytoplasmic, and extracellular components. Incorporation of the stain by elements within each tissue varies from intense to light with a subtle gradation of intermediate shades of purple and blue tones. For most cell structures the density of the stain parallels the electron density of that structure as seen under the electron microscope. For example, nucleoli and heterochromatin stain in dark purple while euterochromatin appear in a light blue shade. In all cases, the embedding media remains unstained. The bond between Stevenel's Blue and the tissues is stable, remaining unaltered by the mounting medium. It is also resistant to time-fading.

Animals↗
PubMed1984

Spontaneous autoradiographic grain activation associated with mast cells in methacrylate plastic embedded tissue sections.

Autoradiographic tracing using tritium labeled compounds or cells is a common laboratory technique for light and electron microscopy. This report describes a chemographic effect associated with certain cells in sections from tissues embedded in the new methacrylate plastic embedding compounds. When tissue sections from rats and rhesus monkeys that received no radioisotope were coated with nuclear track emulsion and subsequently developed, cells with morphologic characteristics of mast cells showed significant grain formation over the entire cell. Three different types of methacrylate plastics were tested using rat and monkey tissues and all three were found to promote grain formation over mast cells; however, this phenomenon was not seen in similar tissue sections from paraffin or epoxy embedded material. The properties of methacrylate plastics which promote positive chemography by mast cells may reflect the greater permeability of this class of plastics. Due to their wide tissue distribution, the presence of such chemographically active cells could cause false estimates of the distribution of either exogenous radiolabeled cells or radioisotopes within many tissues.

Animals↗
PubMed1981

Polyethylene glycol embedded tissue sections for immunoelectronmicroscopy.

Several methods for tissue embedding in polyethylene glycol (PEG) were compared with regard to their applicability for pre-embedding immunoelectronmicroscopy. Existing embedding procedures gave unsatisfactory results and therefore a modified procedure was developed. This method, consisting of very brief tissue infiltration with PEG 1500, to which 3% water is added, allowed adequate tissue sectioning. Using these sections for preembedding immunoelectronmicroscopical localisation of glucagon in bovine pancreatic islets adequate ultrastructural morphology was obtained in combination with excellent preservation of peptide hormone immunoreactivity.

Animals↗
PubMed1992

Simultaneous assessment of TGFB and cell cycle kinetics using IUdR/BrdU infusions in human neoplasms from plastic-embedded tissue.

We describe an immunohistochemical technique that makes use of two monoclonal antibodies (MAb), one to detect the transforming growth factor B (TGFB) and another that reacts with iodo- and bromodeoxyuridine. The purpose of this technique is to determine the relationship between TGFB expression and the S-phase cells in human tumors. Since both can be distinctly identified in situ from tissue embedded in plastic, in assessment of the geographic orientation of S-phase cells in relation to such factors as TGFB, contiguity to blood vessels, nerve fibers, and macrophages can also be achieved.

Antibodies, Monoclonal↗
PubMed1984

A simple, rapid staining procedure for methacrylate embedded tissue sections using chromotrope 2R and methylene blue.

Sections of tissue embedded in glycol methacrylate can be stained in rapid sequence with solutions of 1% aqueous chromotrope 2R adjusted to pH 3 and 0.1% methylene blue to produce sufficient contrast and cellular detail to permit quick visual inspection and/or photomicrography. Solutions of these stains are simple to prepare and are stable over long periods. Staining of sections may be accomplished within six minutes.

Animals↗
PubMed1990

Haemopoiesis in human fetal and embryonic liver. Immunohistochemical determination in B5-fixed paraffin-embedded tissues.

Paraffin embedded tissue reactive monoclonal antibodies were used to study human embryonal and fetal haemopoiesis, combining optimal morphology with immunohistological determination of haemopoietic cell subtypes and their microenvironment. Seven embryonal and twelve fetal liver specimens were studied, having been fixed in B5-fixative and embedded in paraffin. The different haemopoietic lineages each showed their own immunophenotype and distribution; intercellular and microenvironmental relationships were easily determined. Erythroid cells are reactive with VIE-G4, LN1, and MT1, sometimes partly surrounding a central macrophage. Myelomonocytic cells react with LCA, MT1, MB3, LN2, and anti-lysozyme, and from 14 weeks onwards with LN3. Lymphoid cells show LCA, MT1, MT2, MB1, MB2, MB3, and LN2 reactivity. In a few cases some scarce My10+ early progenitor cells were seen. An important finding is the extensive MT1-reactivity distributed over all haemopoietic lineages, and the demonstration of immature haemopoietic blast cells exclusively expressing the MT1 antigen. Further studies employing MT1 are necessary to delineate the extent of the distribution and the possible function of the antigen. Use of the MT1 mAb may contribute to the elucidation of the exact nature of the haemopoietic blast cells and their place in haemopoietic development.

Antibodies, Monoclonal↗
PubMed1991

A comparison of the atomic composition of siderosomes (haemosiderin) in cryosections of unfixed frozen tissues and Epon-embedded tissues.

Siderosomes (i.e. single membrane-bound lysosomal bodies containing haemosiderin) were produced in the liver and muscle of rats by injections of iron dextran. Electron-probe X-ray analysis was executed on siderosomes in cryosections of quick-frozen fresh unfixed tissues (liver and muscle) and sections of Epon-embedded tissues. There were no statistically significant differences between the ratios of iron:phosphorus and iron:sulphur in these two types of preparations. Hence it is concluded that there is no significant loss or gain of phosphorus or sulphur during preparation of tissues for Epon embedding. The results confirm past belief that so little phosphorus or sulphur is present in siderosomes that haemosiderin is best regarded as ferric hydroxide oxide. A new finding in the present study was the demonstration of small amounts of potassium in siderosomes in cryosections. It seems that potassium is lost (leaches out) from siderosomes during preparation of tissues for Epon-embedding.

Animals↗
PubMed1985

Immuno gold staining (IGS) for electron microscopical demonstration of glial fibrillary acidic (GFA) protein in LR white embedded tissue.

Post-embedding immunocytochemical staining methods using gold have so far failed to label intermediate filament antigens in situ in epon or araldite embedded tissue. We have now applied the post-embedding immuno gold staining (IGS) technique for LR White embedded tissue. Glial fibrillary acidic (GFA) protein immunoreactivity was clearly demonstrated electron microscopically on astrocytic filaments of rat cerebellum in situ.

Animals↗
PubMed1989

A modified Brown and Hopps stain for identification of gram-positive and gram-negative microorganisms in glycol methacrylate-embedded tissues.

Plastic embedding is a recently developed technique that has been shown to be superior to conventional paraffin embedding in the histopathologic identification of microorganisms in tissue specimens. This report describes a modification of the Brown and Hopps stain for the differentiation of gram-positive and gram-negative microorganisms in 3-micron-thick plastic sections. This technique is easily performed and has a rapid turnaround time. This staining method permits a more precise histopathologic diagnosis of infectious agents than is possible in paraffin-embedded tissues.

Acrylates↗
PubMed1991

A monoclonal antibody to human leukocyte common antigen, SHL-1, and its use for formalin-fixed, paraffin-embedded tissues.

The authors describe a newly characterized murine monoclonal antibody to the human leukocyte surface antigen, SHL-1. The antigen belongs to the leukocyte common antigen (LCA) family, and its molecular weight is about 180,000 daltons, which is similar to that of some previously characterized LCAs. The SHL-1 antigen is resistant to conventional tissue-fixation and embedding procedures. This antibody can therefore be used in the immunohistochemical staining of paraffin-embedded tissue sections. Wide screening with a sufficient number of both fresh and routinely processed paraffin-embedded tissues was done with indirect immunoperoxidase technique. With this procedure, SHL-1 labeled the majority of normal leukocytes and hematopoietic malignancies. Some B-cell malignancies were not stained with this antibody. The non-hematologic malignancies posing diagnostic problems of differentiation from lymphomas or leukemias were completely negative to SHL-1. The immunoreactivity to SHL-1 of samples from 24 leukemic patients and 15 human tumor cell lines was determined by the immunofluorescence method. Of 24 leukemic preparations, 23 were strongly reactive to this antibody. One case of B-cell leukemia did not react with SHL-1. No immunoreactivity was demonstrated in non-hematopoietic tumor cell lines. The overall reaction pattern of SHL-1 proved its usefulness in both diagnostic and research practice in hematological disorders. This antibody detected cell surface antigens of the T cell series more effectively than those of the B-cell series in terms of the positive number of cells and mean fluorescence intensity.

Antibodies, Monoclonal↗
PubMed1987

The formaldehyde-fixed and paraffin-embedded tissues for diagnostic transmission electron microscopy: a retrospective and prospective study.

The fine structures of tissues processed routinely for light microscopy were studied retrospectively in 44 tissues and tumors and prospectively in 13 tissues and tumors. In the prospective study, we fixed tissues in an ample amount of fixatives, carefully avoiding crushed and air-dried portions, and processed them by five methods. The fine structures of the retrospective cases were mostly poor or passable, whereas those of the prospective cases were generally good. All formaldehyde-fixed tissues showed varied degrees of tissue extraction, notably of lipids, membranous structures, ribosomes, glycogen, and other loose cellular and intercellular matrix materials, that was related to the status and type of tissue as well as the kind and duration of fixation, dehydration, and tissue clearance. Paraffin embedding per se appeared to cause little alteration of the fine structure. Transmission electron microscopy of the paraffin-embedded tissue appeared most useful to identify infective agents, foreign particles, and densely packed organelles or structures, usually in the differential diagnosis of neoplasms. Although many factors are difficult to control, initial careful thin slicing, judicious selection, and fixation of the tissue (even by regular phosphate-buffered formaldehyde solution) can improve the fine structure of paraffin-embedded tissues and be useful in the direct correlation of light and electron microscopic findings.

Bone Marrow Diseases↗
PubMed1986

Ia antigens in plastic-embedded tissues: a post-embedding immunohistochemical study.

The aim of the present study was to establish a plastic embedding technique that makes possible the immunohistochemical demonstration of class II major histocompatibility complex (MHC) antigens (Ia antigens) in undecalcified joint tissues. Therefore a series of fixatives and dehydrating agents was tested for saving Ia immunoreactivity by post-embedding immunostaining of thin sections (2 microns) of rat tissues that had been embedded in glycol methacrylate (GMA), and by comparing with cryostat sections. An indirect immunoperoxidase and the avidin-biotin complex (ABC) technique were used. Combined with fixation by 4% formaldehyde, dehydration with GMA was found to give the best preservation of Ia antigenicity, followed by dehydration with ethylene glycol. The thinness of tissue sections facilitated the association of Ia antigens with different subcellular compartments in distinct cell populations. These various patterns are described.

Animals↗
PubMed1989

Measurement of S-phase fractions in lymphoid tissue comparing fresh versus paraffin-embedded tissue and 4',6'-diamidino-2 phenylindole dihydrochloride versus propidium iodide staining.

S-phase fractions for 62 lymphoid biopsies were calculated, by means of flow cytometry, from both fresh and paraffin-embedded tissue. The purposes of this study were to determine whether significant differences were seen between S-phase estimates obtained from fresh and fixed tissue and to compare results obtained with two DNA dyes, namely 4'-6'-diamidino-2 phenylindole dihydrochloride (DAPI) and propidium iodide (PI). The 62 cases consisted of 38 cases of non-Hodgkin's lymphoma (NHL), 19 reactive samples, and 5 cases of Hodgkin's disease. Fifty-four of the samples showed DNA diploid profiles. A good agreement between S-phase results from fresh and fixed tissue was seen, with technical factors accounting for around 20% of the total variance. Using a paired t test, no significant difference was seen between fresh and fixed tissue for diploid cases, but there was a trend for S-phase estimates from fixed tissue to be higher. If all cases (including the eight DNA aneuploid samples) were included in the analysis this difference just reached statistical significance (P less than .05). In a subgroup of 19 of the cases, a comparison was performed on both fresh and fixed tissue of staining with DAPI and PI. A good agreement between results with both DNA stains was found on fresh and fixed tissue, with no significant differences being apparent, and stain-related factors accounted for only 10% of the total variance.

Cell Cycle↗
PubMed1992

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.

Antibodies, Monoclonal↗