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A method for the demonstration of nonspecific esterase activity in plastic-embedded tissues using Meldola Blue is described. Although Meldola Blue does not function as an electron carrier in this method, it may account for the short incubation time and excellent localization of the reaction product. Use of Meldola Blue is an advancement in the demonstration of enzyme activity in plastic sections.
This report presents a method which can be used for counterstaining semithin sections of plastic embedded tissue. The sections are treated with a solution of silver lactate, followed by physical development. During the silver lactate treatment, silver ions are bound by various tissue components as metallic silver or silver sulfide. During physical development catalytic reduction of silver ions to metallic silver takes place where silver has been bound in the tissue, enlarging the silver deposits to microscopically visible dimensions. The amplified silver deposits give high contrast staining in yellow, brown and black suitable for both color and monochrome photography. The localization of the silver deposits is highly specific and may reflect several independent chemical processes. Examples in several tissues are shown.
Three methods are evaluated for holding capsules of plastic-embedded tissue for rotary microtomy. Use of a V-block is rapid but deforms the capsule. Gluing the capsule to a supporting block is useful for reorientation of the capsule but otherwise time consuming. adapter is easy to use, does not deform the tissue, and is the preferred method for routine microtomy.
In situ hybridization techniques have rapidly become widely used by the molecular biologist for the localization of specific nucleic acid sequences in individual cells or tissues. We describe the demonstration of Sox gene mRNA in chick tissue that has been embedded in the plastic methyl methacrylate to permit the preparation of sections for high-resolution light microscopy. Polymerization of the plastic was induced by using either N,N-dimethylaniline or N,N-3,5-tetramethylaniline. The in situ hybridization technique used was non-isotopic and used a digoxigenin-labelled probe detected with an antibody bound to alkaline phosphatase, which was then localized using X-phosphate-Nitro BT as a substrate-chromogen mix. Various pretreatments of the tissue sections were investigated, including the use of proteinase K, and heat-mediated techniques using a microwave oven and a pressure cooker. The best results were produced using pressure cooking on tissue in which the plastic had been chemically polymerized with N,N-3,5-tetramethylaniline. For the demonstration of Sox 11, this combination had a critical influence on the staining results, but for Sox 21 all protocols used produced good staining.
A procedure is described in which thick sections (2-10 mu or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, alkalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pre-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.
Iron pyronine Y and Alcian blue demonstrated sulfated and nonsulfated acid mucin, respectively, in plastic-embedded sections. Safran used in combination with these two dyes stained collagen and some reticulum fibers. Sulfated acid mucin was red, while nonsulfated acid mucin stained blue; collagen appeared yellow to greenish yellow. The iron pyronine Y-Alcian blue-safran staining method, when used as in the present article, produces excellent cellular visualization of sulfated and nonsulfated acid mucin.
To eliminate sectioning artefacts, we devised a method for the preparation of surfaces of plastic embedded hard tissues and their examination by incident light fluorescence microscopy. Flat surfaces produced by cutting on a microtome or using a low-speed saw were stained with dyes in aqueous solutions. Best results were obtained in tissues embedded in glycol-methacrylate. This polymer allowed good penetration of the dyes and its optical properties render cutting defects invisible with the present method. The relationship between hard and soft tissues was well preserved and the integrity of the mineralized component maintained. Cellular details and the distinction between osteoid and bone could be clearly/demonstrated. The procedure is simple, rapid and produces good results even in instances when histological sectioning is unsatisfactory.
0.5 mu plastic-embedded sections, obtained from aldehyde-osmium fixed rat and cat spinal cord, were impregnated in a 0.5% Protargol solution for 24 hours at 56 degrees C. Reduction was performed in a sodium sulphite- (5%) hydroquinone (1%) developer. Terminals undergoing Wallerian degeneration stand out as easily discernible black dots; corresponding osmiophilic degenerative patterns are demonstrated in consecutive thin electron microscopic sections. This simple technique enables successful trimming of blocks to obtain areas with the highest frequency of terminal degeneration.
Although the utility of antigen labeling techniques in frozen tissues is well known, it is generally acknowledged that an improvement in morphologic preservation is desirable. Conventionally processed paraffin-embedded tissues are limited in the range of antigens that can be detected and newer plastic embedding techniques have been even more restricted. By using cold (4 degrees C) processing and limited fixation a wide range of antigens (including T and B markers) has been demonstrated in 2 mu plastic sections. The morphologic preservation and antigen localization are superior to other techniques. The combination of precise antigen localization and excellent morphologic preservation should expand the diagnostic and investigative uses of immunohistology.
In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semithin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na2 CO3, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.
Hairy-cell leukemia is a lymphoid leukemia of B-cell lineage, the cells of which are characteristically tartrate resistant acid phosphatase positive on blood and bone marrow smears. However, because hairy-cell leukemia is frequently associated with abundant marrow stroma, dry marrow taps, and peripheral pancytopenia, the diagnosis may rest on the appearance of the bone marrow biopsy specimen alone. The ribosome-lamella complex has been associated with hairy-cell leukemia, and can be visualized by light microscopy using l-micron sections of plastic-embedded bone marrow specimens stained with toluidine blue. We describe the findings in a case in which bone marrow and liver biopsy specimens were positive for hairy cells containing ribosome-lamella complex, which were visualized with both electron microscopy and light microscopy. Reliable light microscopic identification of ribosome-lamella complex may provide an easy and inexpensive method of aiding in the diagnosis of hairy-cell leukemia when aspirate material is not available for tartrate-resistant acid phosphatase stain.
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Using a new technique for antigen localization, we have demonstrated platelet proteins in megakaryocytes in plastic-embedded biopsy specimens of normal human bone marrow. In a series of 25 specimens, megakaryocytes showed labeling with antibodies to the integral membrane glycoproteins IIIa, IIb, and the IIb-IIIa complex; granule membrane protein 140; and five alpha-granule matrix proteins: thrombospondin, factor VIII-related antigen, beta-thromboglobulin, platelet factor 4, and fibrinogen. The antibodies to the membrane glycoproteins IIIa, IIb, and IIb-IIIa produced diffuse cytoplasmic staining and heavier staining on the plasma membrane, whereas the antibodies to the alpha-granule matrix proteins produced a distinct granular staining within the cytoplasm. Staining for granule membrane protein 140 was also granular in distribution. Rare mononuclear cells consistent with megakaryocyte precursors were labeled with these markers. Other enzyme histochemical and lectin-binding studies showed that the enzyme alpha-naphthyl acetate esterase, the lectin Ulex europaeus I, and the periodic-acid Schiff reaction were consistent, but not specific, markers of megakaryocytes. This immunohistochemical technique should facilitate the examination of qualitative and quantitative changes in megakaryocytes in a variety of physiologic and pathologic processes.
A technique is described to detect bromodeoxyuridine (BrdU) incorporated by cells in S-phase, with a monoclonal antibody, using removable plastic embedding and immunogold-silver staining (IGSS). The incubation times were reduced and the immunological reactions enhanced by microwave irradiation. The embedding in methyl methacrylate enabled us to make thinner sections and it improved the quality of the preparations. The methyl methacrylate did not hinder the reaction of BrdU with the antibody because it could be removed prior to the IGSS procedure. The IGSS procedure appeared to be very sensitive, requiring lower concentrations of the antibodies than other methods. The use of microwave irradiation shortened the time needed to stain a section from 7 to less than 4 h. Furthermore, using microwave irradiation, the concentration of the antibodies needed could be reduced even further compared with the normal IGSS procedure. In sections of the mouse testis and small intestine only nuclei of cells known to be able to proliferate appeared BrdU positive. The non-specific background staining was found to be negligible. In testes of mice that received both 3H-thymidine and BrdU more than 95% of the radioactively labelled cells also showed BrdU label and vice versa. This indicates that both methods are equally sensitive for detecting cells in S-phase.
The preparation of a set of aminoplastic standards for quantitation of sodium, magnesium, phosphorus, sulfur, chlorine, potassium, and calcium in ultrathin plastic sections of soft tissue is described. The standards are low in cost and easy to prepare and handle. They cover concentrations up to 750 mmoles/kg dry weight, and display chemical and physical properties similar to those plastic-embedded samples. The standards can be used to convert peak-to-continuum ratios obtained from the specimen to elemental concentrations. An application of these standards is shown using rat exocrine pancreas as a model. The standards can also be used to advantage for the calibration of the detector efficiency.
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Plastic (polymer) embedding is recommended as the method of choice for studying small biopsies such as those obtained from liver, bone marrow, lung and by endoscopy from the gastro-intestinal tract. The method offers the opportunity to examine one to four micron thick sections and, therefore, to evaluate cytologic detail. Multiple levels can be obtained from such fragments, and the reasonably large blocks provide adequate section size for diagnostic purposes.
We describe a new method for light microscopic demonstration of alkaline phosphatase (ALP) activity in plastic-embedded sections. Rat tissues were fixed in acetone (-20 degrees C), infiltrated in glycol methacrylate (GMA), and embedded at 0 degrees C. Sections were cut at 1 and 2 microns, dried at room temperature, and incubated in the conventional Gomori medium. Cerium chloride was used to convert calcium phosphate into cerium phosphate, which was subsequently converted into cerium perhydroxide. The slight yellow precipitate of cerium perhydroxide was amplified using 3,3'-diaminobenzidine tetrahydrochloride (DAB). For comparison, tissue sections were processed according to the calcium-cobalt method. The method described combines exact localization of ALP activity with optimal preservation of tissue morphology.