scholarly journals Coloidal gold, ferritin and peroxidase as markers for electron microscopic double labeling lectin techniques.

1978 ◽  
Vol 26 (3) ◽  
pp. 163-169 ◽  
Author(s):  
J Roth ◽  
M Binder
Keyword(s):  
Particle Size ◽  
Cell Surface ◽  
Quantitative Evaluation ◽  
Binding Sites ◽  
Colloidal Gold ◽  
Lectin Binding ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Optimal Amount ◽  
Lectin Binding Sites

Three markers, colloidal gold, ferritin and peroxidase, were checked for usefulness in double labeling of lectin-binding sites. The amount of various lectins for the stabilization of good sols of a different particle size was evaluated. Several lectin-gold complexes were prepared for electron microscopic labeling purposes, and the optimal amount of various lectins needed for stabilization of gold solutions of a different particle size was determined. The following combinations were investigated for their usefulness in labeling two different lectin-binding sites: lectin-gold and lectin-gold (different particle size), lectin-gold and lectin-ferritin, as well as lectin-ferritin and lectin-peroxidase. Of these combinations the latter did not give satisfactory results for double labeling. In all single and double labeling techniques with the above mentioned markers the quantitative evaluation of the number of lectin-binding sites is not feasible, but these techniques will be of considerable value for the investigation of the dynamics of different lectin-binding sites on the cell surface.

scholarly journals Hepatocyte cell surface polarity as demonstrated by lectin binding.

1988 ◽  
Vol 36 (12) ◽  
pp. 1561-1571 ◽  
Author(s):  
P N McMillan ◽  
D C Hixson ◽  
K A Hevey ◽  
S Naik ◽  
H O Jauregui
Keyword(s):  
Cell Surface ◽  
Binding Sites ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Separation Procedure ◽  
Lectin Receptors ◽  
Mechanical Methods ◽  
Dissociated Cells ◽  
Lectin Binding Sites

We performed an investigation at the ultrastructural level of the differential distribution of lectin-binding sites among sinusoidal, lateral, and bile canalicular domains of adult rat hepatocytes. Lectin binding to hepatocyte glycocalices was studied in situ or after cellular dissociation by enzymatic (collagenase), chemical (EDTA), and mechanical methods, as well as during cell culture. Using thirteen biotinylated lectins and an avidin-biotin-peroxidase complex (ABC), we have identified lectin-binding sites that are predominantly localized in the bile canalicular [Ricinus communis agglutinin (RCA)] or sinusoidal [Phaseolus vulgaris (PHA)] domains in situ and in mechanically dissociated cells. Lens culinaris (LCA) staining was prominent on sinusoidal surfaces, slight along lateral surfaces, and completely absent in the bile canalicular domain. Concanavalin A (ConA) was unique in binding equally to all domains. Triticum vulgaris [wheat germ agglutinin (WGA)] was also bound to all domains, but most intensely to the bile canalicular region. Cells dissociated via collagenase or EDTA treatment exhibited a spherical morphology characterized by many surface microvilli and absence of morphological domains. Lectin binding to dissociated cells was uniformly distributed over the entire cell surface, suggesting a redistribution of lectin receptors that was independent of the separation procedure. Hepatocytes in culture exhibited a partial restoration of morphological domains, but lectin binding polarity was not re-established.

scholarly journals Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes.

1983 ◽  
Vol 96 (5) ◽  
pp. 1189-1196 ◽  
Author(s):  
J Roth ◽  
D Brown ◽  
L Orci
Keyword(s):  
Basement Membrane ◽  
Cell Surface ◽  
Binding Sites ◽  
Wheat Germ ◽  
Lectin Binding ◽  
Glomerular Cell ◽  
Thin Sections ◽  
Foot Process ◽  
Wheat Germ Lectin ◽  
Lectin Binding Sites

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold-particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin-binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.

scholarly journals Peroxidase and gold complexes of lectins for double labeling of surface-binding sites by electron microscopy.

1977 ◽  
Vol 25 (10) ◽  
pp. 1181-1184 ◽  
Author(s):  
J Roth ◽  
M Wagner
Keyword(s):  
Electron Microscopy ◽  
Horseradish Peroxidase ◽  
Concanavalin A ◽  
Binding Sites ◽  
Lectin Binding ◽  
Helix Pomatia ◽  
Surface Binding ◽  
Double Labeling ◽  
Membrane Bound ◽  
Lectin Binding Sites

Double labeling experiments were performed for visualization of the binding sites of Concanavalin A and anti-AHel (the lectin from Helix pomatia). The anti-AHel was labeled with the colloidal gold whereas the membrane bound Concanavalin A was demonstrated by an affinity technique using horseradish peroxidase. The two markers used could be clearly distinguished electron microscopically. The specificity of the cell surface double labeling was demonstrated in the control experiments. A topological distinct localization of the both lectin-binding sites is evident.

scholarly journals Applications of immunocolloids in light microscopy. II. Demonstration of lectin-binding sites in paraffin sections by the use of lectin-gold or glycoprotein-gold complexes.

1983 ◽  
Vol 31 (4) ◽  
pp. 547-552 ◽  
Author(s):  
J Roth
Keyword(s):  
Binding Sites ◽  
Colloidal Gold ◽  
Rat Kidney ◽  
Lectin Binding ◽  
High Specificity ◽  
Step Method ◽  
Gold Complexes ◽  
Tissue Sections ◽  
One Step ◽  
Lectin Binding Sites

A new procedure is presented for the light microscopic demonstration of specific sugar sequences of oligosaccharides in glycoconjugates by lectins combined with the colloidal gold marker system. Tissue sections from aldehyde-fixed and paraffin embedded rat kidney were stained either in a one-step method with lectin directly bound to particles of colloidal gold or in a two-step method using non-labeled lectin and glycoprotein labeled with colloidal gold. In both methods the presence of lectin-binding sites in the tissue sections is revealed by the appearance of a red coloration that is due to the accumulation of gold particles. The high specificity of the technique is combined with a good sensitivity and resolution as demonstrated by a differential plasma membrane staining in renal epithelial cells. The lectin-gold or glycoprotein-gold complexes remain stable for months and produce a permanent nonbleaching staining.

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