Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry.
In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold has bound to the antigens in the cells. We investigated several aspects of gum arabic-silver lactate-hydroquinone enhancement solution (Danscher solution) by examining gold-conjugated antibodies embedded in agar, sectioned on a vibrotome, and enhanced with different solutions. The rate of silver enhancement was optimized in 50% gum arabic and 200 mM HEPES buffer, pH 5.8. We also examined chemicals used as developers and found that N-propyl gallate (NPG) gave a more uniform development than the routinely used hydroquinone (HQ). The diameter of the silver-enhanced particles after incubation in osmium tetratoxide (OSO4) decreased somewhat with longer incubation time and higher percentages, but the density (number per unit area) of silver-enhanced particles was little changed. The loss of silver-enhanced particle diameter was reduced by lowering the concentration of OSO4 to 0.1%. Comparison of commercial small gold probes showed that NPG enhancement of Nanogold gave more uniform particle size and a better correlation between enhancement time and particle density. When this procedure was applied to cell cultures with monoclonal antibodies, the silver-enhanced particles were similar to those in the agar sections. When free-floating tissue sections were used, longer silver enhancement times were needed to obtain similarly sized particles. This new NPG-silver-enhancement procedure offers a reliable and easy method to localize proteins in cultured cells and tissue sections by pre-embedding electron microscopic immunocytochemistry.
Burry
R W
RW
Department of Cell Biology, Neurobiology and Anatomy, Ohio State University, Columbus 43210-1239.
Vandré
D D
DD
Hayes
D M
DM
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D002478:Cells, Cultured; D002531:Cerebellum / Q000737:chemistry / Q000166:cytology; D006046:Gold; D006170:Gum Arabic; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*; D009992:Osmium; D011435:Propyl Gallate; D051381:Rats; D012834:Silver
D000906:Antibodies; D009992:Osmium; D012834:Silver; D006046:Gold; D011435:Propyl Gallate; D006170:Gum Arabic