Plastic-embedded semi-thin sections of fine needle aspiration biopsies with dibasic staining. Diagnostic and didactic applications

The diagnostic and didactic utility of plastic-embedded semi-thin sections of fine needle aspiration biopsies is presented using a case-study approach. The Spurr epoxy semi-thin sections were stained with a newly developed sequential basic fuchsin-methylene blue stain, which gives hematoxylin-and-eosin-like staining and simultaneously substitutes for a wide variety of special stains. The informational content of the sections can approach that of electron microscopy. The use of a direct off-the-slide "pop-off" technique in preparing the plastic-embedded sections allows for a direct comparison between similar groups of cells embedded in plastic and present on the routine aspiration slides; retrospective analysis can discern subtle, previously unrecognized morphologic features in the alcohol-fixed, Papanicolaou-stained slides. The limitations of this comparative approach, however, become manifest when the effects of alcohol fixation on cells are directly compared in plastic and at the ultrastructural level to aldehyde fixation.     

Immunogold silver staining associated with epi-fluorescence for cucumber mosaic virus localisation on semi-thin sections of banana tissues

The immunogold-silver staining (IGSS) technique in combination with epi-fluorescence detection was used to localise cucumber mosaic virus (CMV) particles within banana infected tissues. For this purpose, tissue samples (2 mm3) were excised from CMV-infected and highly proliferating meristem cultures of Williams BSJ banana (ITC. 0570, AAA, Cavendish subgroup). These samples were immediately fixed in a 2% paraformaldehyde/0.25% glutaraldehyde mixture, dehydrated in ethanol, and finally embedded in L.R.White resin. Semi-thin sections were cut, mounted on clean treated glass slides and immunostained for CMV particles using gold-labelled secondary antibodies and silver enhancement. Sections were counterstained with basic fuchsin and examined using laser scanning confocal microscopy. Negative controls included immuno-stained samples excised from non-virus infected material as well as infected material on which primary or secondary antibodies were not applied. Images of autofluorescence (in red) and of epi-reflectance of silver-enhanced immunogold particles (in green) were recorded separately and merged, allowing the specific localisation of CMV particles at the cellular level on semi-thin sections of aldehyde-fixed banana tissues. The main advantage of this analytical approach compared to previously published protocols is that it combines a fast staining procedure, stable preparation, a high resolution, and a narrow plane of focus with the flexibility in generation, processing and analysis of images offered by laser scanning confocal microscopy. Finally, the presence of numerous CMV particles within banana meristems constitutes a clear explanation of the very low CMV elimination efficiency when using meristem-tip culture alone.     

Cement line staining in undecalcified thin sections of cortical bone

A technique for demonstrating cement lines in thin, undecalcified, transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Cement line staining in undecalcified thin sections of cortical bone.

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

Microwave-stimulated staining of plastic embedded bone marrow sections with the Romanowsky-Giemsa stain: improved staining patterns

Staining plastic sections with the Romanowsky-Giemsa method is both time-consuming and difficult. This paper reports how the staining time can be reduced to 25 min using microwave irradiation of the staining solution. It is shown that staining results depend on the fixative used, staining temperature, dye concentration and pH of the staining solution as well as on several parameters of the microwave irradiation technique. The staining patterns are improved when compared with those obtained by conventional staining of plastic sections. The colors are more brilliant and greater contrasts are observed. Basophilia, polychromasia, and orthochromasia accompanying red cell maturation are more pronounced. For white cell maturation the initial appearance of specific granules (neutrophil, basophil, and eosinophil) is more evident. Thus, cell classification is easily accomplished using the described technique. It is suggested that microwave-stimulated staining be considered for routine use.     

Surface staining of sawed sections of undecalcified bone containing alloplastic implants.

A simple new method is described for the histological evaluation of bones containing alloplastic implants of ceramic and/or metallic materials. The undecalcified bone is embedded in acrylic resin and sectioned at 50-200 micrometer using a sawing microtome. One surface of the preparation is stained up to 10 micrometer deep by floating the preparation on Giemsa stain. Other staining procedures are possible. Microscopic detail is satisfactory for histological and morphometric evaluation.     

Immunocytochemical localization of plasmalemmal proteins in semi-thin sections of epithelial monolayers

We describe a technique for analysis by light microscopic immunocytochemistry of the distribution of plasmalemmal proteins in polarized epithelial cells. For this purpose, Madin Darby Canine Kidney (MDCK) cells were grown to confluency on Cytodex beads, the beads were fixed with formaldehyde, and semi-thin (0.5 micron) sections were cut at liquid nitrogen temperature on an ultracryomicrotome. The distribution of the basolaterally distributed plasmalemmal protein, Na,K-ATPase, was assessed by indirect immunofluorescence using a monospecific polyclonal antibody directed against the alpha-subunit of the Na pump. Such preparations enable epithelial monolayers to be evaluated in cross-section, thus permitting unambiguous topological assessment of apical and basolateral membrane proteins. Thus, the spatial uncertainties encountered in en face examination of membrane protein distribution in epithelia grown on solid supports are largely obviated. In addition, we describe a technique for removal of the bead matrix, which markedly reduces nonspecific background staining and improves access of reagents to the basal cell surface, thus permitting localization of basal lamina components.     

Alternative staining methods for Lowicryl sections.

A number of stains and stain combinations have been identified that, when used with the hydrophilic resin Lowicryl K11M, produce marked improvements over aqueous uranyl and lead salts (UA-Pb) in terms of low granularity, specificity, and range of components contrasted. Three test specimens, tobacco mosaic virus (TMV), starfish sperm, and cultured mouse fibroblasts, were used to evaluate stain characteristics. UA-Pb showed a preference for nuclei acids, which were stained specifically by osmium ammine-B at pH 1.5. A number of stain combinations in which UA was followed or preceded by salts containing barium, manganese, tungsten, molybdenum, and vanadium provided excellent staining of protein-containing components, each stain combination being unique in terms of the degree to which specific components were discriminated. These stains were particularly effective for visualizing internal components of the nucleus where a number of fibrillar and particulate structures not seen with UA-Pb were well contrasted.