Ultrastructural visualization of cellular carbohydrate components by means of lectins on ultrathin glycol methacrylate sections.

A method for the visualization of cellular carbohydrate components by both light and electron microscopy using lectins on glycol methacrylate sections is proposed. This method, which is an application of the lectin-peroxidase affinity technique, solves the problem of limited penetration when it is attempted to demonstrated lectins receptors within the tissue block. Following partial dissolution of glycol methacrylate from thin sections using alcohol, they are incubated successively with lectin (Concanavalin A or wheat germ agglutinin), horseradish peroxidase (Sigma, type II), 3-3' diaminobenzidine and H2O2 and then with OsO4-Different kinds of tissues and cells have been used to test the method: mouse myocardium, rat epididymis, a protozoon Gregarina blaberae and the bacterium Escherichia coli. The localization of carbohydrate residues deomonstrated by this method within the different tissues and cells is consistent with the findings from other published studies. Controls have been performed (i.e., omission of the lectin, lectin and its inhibitor) and these demonstrate the specificity of the method.     

Ultrastructural visualization of sites binding concanavalin a on the cell membrane ofDaucus carota

Summary Protoplasts fromDaucus carota showed differences in binding to Concanavalin A according to which of three enzyme preparations tested were used for their isolation. Protoplast bound Concanavalin A was visualized ultrastructurally using a peroxidase-diaminobenzidine staining. The variable amount of staining was supposed to be due to differences in the composition of contaminating membrane active enzymes in the crude enzyme preparations. Enzymatically removed binding sites for Concanavalin A in the plasmalemma were rapidly resynthesized when the isolated protoplasts were incubated in a sorbitol solution. At room temperature, Concanavalin A bound to the plasmalemma was found in clusters, while at 4 °C and on prefixed protoplasts the binding sites were homogeneously distributed. These results and the effects of the crude enzyme preparations on the cell membrane of the protoplasts will be discussed in relation to the fluid membrane model.     

A dual staining method for neutral complex carbohydrates using alkaline phosphatase-labeled concanavalin A and periodic acid-Schiff

A method has been developed for the dual staining of neutral complex carbohydrates in light microscopy. It combines an alkaline phosphatase-labeled concanavalin A-5-bromo-3-indolyl phosphate, p-toluidine salt (Con A-ALP-BIPT) method with periodic acid-Schiff (PAS) sequence. With the present dual staining method, it is possible to color alpha-D-glucosyl and alpha-D-mannosyl residues blue and 1,2-glycol groups of neutral complex carbohydrates magenta. The validity of this method has been confirmed with appropriate histochemical controls and enzyme digestions on test tissues.     

A dual staining method for neutral complex carbohydrates using alkaline phosphatase-labeled concanavalin a and periodic acid-Schiff

Summary A method has been developed for the dual staining of neutral complex carbohydrates in light microscopy. It combines an alkaline phosphatase-labeled concanavalin A-5-bromo-3-indolyl phosphate, p-toluidine salt (Con A-ALP-BIPT) method with periodic acid-Schiff (PAS) sequence. With the present dual staining method, it is possible to color -d-glucosyl and -d-mannosyl residues blue and 1,2-glycol groups of neutral complex carbohydrates magenta. The validity of this method has been confirmed with appropriate histochemical controls and enzyme digestions on test tissues.     

Ultrastructural evidence of maintenance of concanavalin A binding sites in enucleated mammalian cells

Mouse L cells were enucleated by centrifugation in cytochalasin B. Following enucleation, the enucleated cells were incubated in fresh medium for 30 min, 4, 20, or 24 h before being fixed for electron microscopy. After fixation the cells were incubated in concanavalin A and then horseradish peroxidase was bound to the ConA. Electron microscopy of these enucleates revealed that the concanavalin A-binding sites (CABS) are present on the cell surface of the enucleates even at 24 h after enucleation. Although the method does not detail the number of sites present, the inherent distribution of sites appears similar in normal and enucleated cells. Furthermore, the sites are still functional in that the live enucleated cells are agglutinated by ConA to the same extent as are normal L cells—about 80% agglutination in each instance. The results of this study indicate that surface CABS are maintained in the absence of a nucleus and they are still present even after the Golgi apparatus is morphologically disrupted. Turnover of these sites and their relationship to nuclear function are discussed.     

Ultrastructural visualization of selective peanut agglutinin binding sites in rat osteoclasts

We investigated the distribution of concanavalin A (ConA)-reactive alpha-D-mannosyl and alpha-D-glucosyl groups and peanut agglutinin (PNA)-reactive beta-D-galactose-(1----3)-N-acetyl-D-galactosamine residues on the surface of osteoclasts with pre-embedment ultrastructural lectin cytochemistry after aldehyde fixation of the metaphyses of the rat tibiae. By routine morphology, the plasma membrane of the ruffled border of the osteoclast was distinguished from the rest of the cell membrane, with the exception of the membrane of coated pits, by its characteristic thick coat at its cytoplasmic surface. Cytochemistry, using ConA in combination with horseradish peroxidase (ConA-HRP) and PNA conjugated to HRP, showed that binding of ConA was distributed over the entire cell surface of osteoclasts. In contrast, intense binding of PNA was limited to the membranes of the ruffled border and coated pits, whereas the remainder of the cell membrane stained weakly or not at all. These results demonstrate that preferential PNA binding sites of the cell surface correspond to coated membranes associated with osteoclastic endocytosis.     

Ultrastructural localization of Thermoplasma acidophilum surface carbohydrate by using concanavalin A

Surface carbohydrate, presumably the lipopolysaccharide, of Thermoplasma acidophilum was visualized by means of the concanavalin A, horseradish peroxidase, and diaminobenzidine cytochemical staining procedure.     

The application of Miller's elastic stain to glycol methacrylate tissue sections

The application of Miller's dilute elastic stain followed sequentially by Gill's III hematoxylin and a fast green counterstain produced a reliable and consistent method for differentially staining elastic fibers, nuclei, muscle and collagen in glycol methacrylate tissue sections. Evaluation of different methods of fixation and conditions of staining on animal tissue sections showed that elastic fibers in both perfusion and immersion fixed tissues can be intensely stained. The stability of Miller's elastic stain offers the potential of a commercially available histological stain reagent for coarse and fine elastic fibers in glycol methacrylate tissue sections.     

Ultrastructural localization of pituitary hormones by immunocytochemical reactions in ultrathin sections of adenomas

Human pituitary adenomas were fixed in glutaraldehyde and embedded in epon. Ultrathin sections were incubated either with anti-hGH, anti-hPRL or anti-hLH. They were incubated with second step goat anti-rabbit immunoglobulins linked to gold particles. Two PRL secreting adenomas, one GH and PRL secreting adenoma, one ACTH secreting adenoma and two non secreting adenomas were studied. The specificity and the limits of the method were discussed in relation with the results obtained in light microscopy with the PAP method.     

Ultrastructural immunogold labelling of vimentin filaments on postembedding ultrathin sections of arachnoid villi and meningiomas

An immunoelectron microscopic technique for the labelling of vimentin intermediate filaments on postembedding ultrathin sections is reported. Arachnoid villi obtained at autopsy and meningiomas at surgery were fixed in 1% paraformaldehyde for 30 minutes, embedded without postfixation in Epon-Araldite mixture and polymerized at 37 degrees C for 3 weeks. Ultrathin sections were etched in 2% KOH for 3 minutes and incubated with anti-vimentin monoclonal antibodies which were subsequently labelled with goat anti-mouse IgG coupled to colloidal golds. All of these labelling procedures were consistently performed within 4 hours. In both arachnoidal and meningioma cells, immunogolds preferentially decorated the intermediate filaments in proportion to the concentration. Very few gold particles were seen over the nucleus, Golgi zone, mitochondria and the extracellular connective tissue fibres. The present technique may be applied to the immunogold labelling of intermediate filaments on postembedding ultrathin sections.     

Ultrastructural studies of the binding of concanavalin A to the plasmalemma of higher plant protoplasts

Summary The binding of concanavalin A to the plasmalemma of higher plants has been studied using protoplasts of two species. The lectin aggregates both tobacco (Nicotiana tabacum L.) leaf protoplasts and protoplasts prepared from a suspension cell culture of grapevine (Vitis vinifera L.). Differences in lectin binding have been investigated using concanavalin A conjugated to ferritin or bound to colloidal gold. Tobacco protoplasts exhibit continuous and saturated labelling of the plasmalemma surface with gold-concanavalin A mixtures. Vine protoplasts under the same conditions show a discontinuous and patchy distribution of label. These results are discussed in terms of a possible binding mechanism.Abbreviations ConA concanavalin A - PBS Phospholi Buffered Saline - PEG polyethylene glycol