Ultrastructural detection of DNA-incorporated bromodeoxyuridine in resin embedded brain tissue

We describe a protocol for the ultrastructural detection of DNA-incorporated bromodeoxyuridine (BUdR) in resin embedded tissue by means of post-embedding immunogold labeling. The paraventricular zone of rat embryos brains was dissected, fixed either in paraformaldehyde or glutaraldehyde, and embedded in LR White. BUdR gold labeling was only found when thin sections were pretreated with 4 N HCl. Other DNA denaturing agents, such as Na ethoxide, formamide, formic acid, heat or HCl at lower concentrations were ineffective. Very little difference in the degree of labeling was found depending on the fixation. This method can be applied to investigate the fine structure of replicating cells in other in vivo conditions, such as human tumors.     

A simple method using alizarin red S for the detection of calcium in epoxy resin embedded tissue

This report presents a simple procedure for staining 1-2 microns epoxy plastic sections of cells and mineralizing matrix present in fetal bovine bone tissue cultures. A 0.3% aqueous toluidine blue O solution was used as a cellular stain and was followed with 2% alizarin red S for the detection of calcium at sites of mineralization. Effects of concentration and pH of alizarin red S on the penetration of epon embedded thick sections were investigated. Optimal staining was achieved with a 2% aqueous alizarin red S solution adjusted to a pH of 5.5-6.5. This staining procedure provides unusually clear contrast between mineral and bone cells in plastic sections for light microscopy.     

gamma-Glutamyl transpeptidase demonstrated in tissue sections embedded in glycol methacrylate resin

Summary A method is described for the histochemical demonstration of -glutamyl transpeptidase in tissue sections embedded in glycol methacrylate at low temperature. Enzyme activity was preserved by a short (3 h) fixation of tissue in 4% paraformaldehyde at 4° C prior to embedding at 4° C. Tissue embedded in glycol methacrylate combined good morphology with accurate enzyme localization. Blocks of the embedded tissue could be stored at room temperature for at least 3 months without loss of enzyme activity. The resin is non-fluorescent, allowing the use of the fluorescent coupling agent 5-nitrosalicylaldehyde to visualize the reaction product.     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Summary Bromodeoxyuridine (BrdUrd) immunohisto-chemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and ³H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0:05% proteinase at 37°C for 20 min and hydrolysis with 1 N HCl at 37°C for 20 min was sufficient to detect BrdUrd immunore-activity in ³H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd, immunohistochemistry in cell-kinetic studies.     

Ultrastructural localization study of two Wolfgram proteins in rat brain tissue

Brain Cell Biology - The ultrastructural immunohistochemical localization of Wolfgram proteins W1 and W2 is described in young rat brain tissue. The labelling by the antiserum to W1 is restricted...     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Bromodeoxyuridine (BrdUrd) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and 3H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0.05% proteinase at 37 degrees C for 20 min and hydrolysis with 1N HCl at 37 degrees C for 20 min was sufficient to detect BrdUrd immunoreactivity in 3H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd immunohistochemistry in cell-kinetic studies.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8–12 h at –50 to –40°C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45°C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4°C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens.

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8-12 h at -50 to -40 degrees C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45 degrees C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4 degrees C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Gold labeling with wheat germ agglutinin and RNAse on osmicated tissue embedded in epoxy resin

Tissue of an insect, Lucilia cuprina, fixed conventionally in buffered glutaraldehyde and osmium and embedded in epoxy resin (epon or epon/araldite), provided sections which could readily be labeled with RNAse/gold and wheat germ agglutinin (WGA)/gold. This method offers labeling of tissues with improved contrast and allows the retrospective application of RNAse and WGA labeling to conventionally prepared tissues, without recourse to oxidizing/etching agents.     

Electron probe analysis of freeze-substituted,epoxy resin embedded tissue for ion transport studies in plants

Summary A new technique is described to prepare plant material for electron probe analysis. Root segments 1 mm in length were frozen at-170°, freeze-substituted with anhydrous ether at-30° and infiltrated with Spurr's low-viscosity epoxy resin embedding medium at low temperatures. Sections 1 and 2 thick were cut anhydrously using hexylene glycol in the ultramicrotome trough, mounted on the polished surface of a Be disc and vacuum coated with 150–200 Å aluminum.The new technique allows retention of water-soluble ions at the original sites in the tissue and is superior to cryostat sectioning in spatial resolution of electron probe analysis and in the preservation of cellular structures.The lateral transport of K⁺ into the xylem of corn roots has been successfully studied by electron probe analysis of freeze-substituted, epoxy resin embedded material.     

AFM of biological material embedded in epoxy resin

We present a simple method to extract morphological details from the block face of epoxy embedded biopolymers by AFM. It is shown that topographical contrast and the identification of small structural details critically depend on the procedure of sample preparation before embedding (chemical fixation or high-pressure freezing and freeze-substitution) and on the hardness of the embedding epoxy resin. Ethanol treatment of the block face of the sample after microtomy elutes non-cross-linked polymer chains and makes the smallest details of the embedded biomaterial amenable to detection. AFM (height and phase contrast) examination of the block face of accordingly prepared cells of Caenorhabditis elegans provides data that are comparable to TEM.     

Evaluation of immunocytochemical detection methods of incorporated bromodeoxyuridine in hybridomas

Bivariate distributions obtained from nominal acid hydrolysis or thermal treatment methods used in the cell cycle analysis of incorporated bromodeoxyuridine were shown to be unacceptable with hybridomas. Four different cell treatment and staining methods were compared. These methods are acid hydrolysis, thermal denaturation, nuclei extraction with pepsin digestion, and simultaneous pepsin digestion and acid hydrolysis. The nuclei extraction method was determined to be the most appropriate for the immunocytochemical staining of incorporated bromodeoxyuridine in hybridomas. The resulting bivariate distribution provides a clear distinction between labelled and unlabelled cell fractions. The method based on nuclei extraction with pepsin digestion was optimized for a hybridoma line used in this study.     

Immunohistochemical detection of DNA replication in mouse uterine cells by bromodeoxyuridine labeling of wax- and resin-embedded tissue sections.

To apply the bromodeoxyuridine (BrdU) labeling method using a monoclonal antibody to the study of cell proliferation in the mouse uterus, methods of fixation and embedding of tissues and of immunofluorescent staining were compared in terms of the rate of detection of labeled cells and specificity and stability of fluorescence obtained. BrdU was administered intravenously 2 hr before death and uterine blocks were embedded in polyester wax and Technovit resin after fixation in formalin and periodate-lysine-paraformaldehyde, respectively. The indirect method with anti-BrdU and fluorescein isothiocyanate (FITC) conjugated antimouse IgG antisera and the direct method with FITC conjugated anti-BrdU antibody were applied to both wax- and resin-embedded sections. Labeled and total cells were counted in luminal and glandular epithelia and stomata adjoining them. Counterstaining with hematoxylin for counting total cells produced intense fluorescence over the whole of resin sections and made counting of labeled cells impossible. On wax sections, on the other hand, the results were satisfactory, although the number of labeled cells detected was decreased slightly. In wax sections fluorescence due to nuclear incorporation of BrdU in the indirect method could be easily distinguished from the cytoplasmic or extracellular emission seen in some cells by its location and characteristic color. In resin sections, however, more careful observation was needed since the second antibody used in the indirect method cross-reacted with IgG in eosinophils and produced cytoplasmic fluorescence of the same color. By the indirect method greater numbers of labeled cells were detected in wax sections than in resin sections. The difference was distinct in tissues with extensive cell proliferation. By the direct method the fluorescence obtained was weaker and apt to fade more quickly than that obtained by the indirect method; use of the direct method reduced the number of labeled cells detected in both wax- and resin-embedded sections.     

Use of LR white resin for post-embedding immunolabelling of brain tissue.

The object of this study was to investigate the applicability of the acrylic resin 'LR White' to immunolabelling of various antigenic determinants in aldehydefixed rat CNS tissue. Antibodies were used, which worked well in paraffin sections and therefore were suitable to detect antigens resistant to complete dehydration and heat. Different LR White embedding protocols were employed in order to select the preparation conditions that adequately preserved both the antigenicity and fine structure. Specimens were completely dehydrated with up to 100% ethanol, which was followed by various infiltration times with LR White monomer. Polymerization of the resin was induced by heat, a chemical catalytic procedure (accelerator), or ultraviolet (UV) light. Paraffin, as well as semithin and ultrathin LR White sections were incubated with antibodies reacting to antigens located on the cell surface (stage-specific embryonic antigen-1; SSEA-1), within the plasma membrane (myelin basic protein), in the cytosol (HNK-1, S100 protein), in the cytoskeleton (GFAP, vimentin, neurofilament protein, INT-FIL), and in the extracellular matrix (laminin). All of the examined antigens were immunocytochemically detectable in paraffin-embedded material, while the carbohydrate moieties, HNK-1 and SSEA-1, were not immunoreactive in LR White sections. However, in cryostat sections processed for pre-embedding immunoelectron microscopy, the HNK-1 epitope and SSEA-1 were immunolabelled. Polymerization carried out under UV light led to better structural preservation of brain tissue than resin cured with heat or catalyst. The length of prior infiltration with monomer apparently had no effect on tissue preservation. Consequently, UV light-induced polymerization of LR White gives acceptable morphology of brain tissue. However, the use of this acrylic resin is restricted to the detection of some CNS antigens only.     

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

Summary 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.     

Corrections in counting paired nuclei labelled with bromodeoxyuridine in tissue sections

Bromodeoxyuridine is finding increasing use as an alternative to, or in conjunction with, tritiated thymidine for labelling nuclei in DNA synthesis. Precise identification of labelled nuclei is possible, even when there is considerable overlap between neighbouring nuclei. In the sparsely labelled renal cortex of the normal male mouse, 'flash labelling' with bromodeoxyuridine shows single labelled nuclei at 1 h. At 24, 48 and 72 h after injection of bromodeoxyuridine, some labelled cells are seen to lie adjacent and such labelled pairs are presumed to be the result of cell division. Single labelled nuclei at 24, 48 and 72 h might indicate arrest in DNA synthesis or a prolonged G2 period, but it is important to recognize that a correction must be made for paired labelled nuclei in which one member is out of the plane of section. The factors involved in making such a correction are discussed and a correction table calculated. In the normal male mouse renal cortex, we show that nearly all cells labelled at 1 h had divided by 72 h.     

Cell kinetic studies of in situ human brain tumors with bromodeoxyuridine

At the time of surgery, 18 patients with various brain tumors were given a 1-h i.v. infusion of bromodeoxyuridine (BrdUrd), 150-200 mg/m2. At an infusion rate of 200 mg/m2/h, serum BrdUrd levels of 8 microM were achieved. After the infusion, tumor tissue was obtained and divided into two portions. One portion was fixed in 70% ethanol, embedded in paraffin, and sectioned; the sections were deparaffinized, denatured with 2 N HCl, and reacted with monoclonal antibodies against BrdUrd (anti-BrdUrd MAb). BrdUrd-labeled nuclei were demonstrated satisfactorily by an indirect peroxidase method. The other portion was dissociated into single cells with a DNase enzyme cocktail and reacted with FITC-conjugated anti-BrdUrd MAb to determine the percentage of BrdUrd-labeled cells or with chromomycin A3 for DNA analysis. The single-cell suspensions were analyzed by flow cytometry. The fraction of S-phase cells in the tissue sections was similar to both the percentage of BrdUrd-labeled nuclei and the S-phase fraction determined by flow cytometric analysis. The results obtained with BrdUrd-labeled nuclei were similar to those obtained from previous autoradiographic studies of various brain tumors exposed to a pulse of 3H-thymidine. Since BrdUrd is not radioactive and is nontoxic at the dosage used, these techniques, together with the histopathological diagnosis, may help to predict the biological malignancy of individual tumors.