Tissues previously osmicated for electron microscopy must be deosmicated before silver detection can be employed. Osmication does not destroy immunoreactivity of glutaraldehyde-linked free amino acids detected by anti-hapten IgGs, but can restrict the mobility of targets as it is a very rigid, short-range cross-linker. Traces of osmium are spontaneous sites for silver deposition during the enhancement procedure and must be removed. The time required for removal is dependent on the amount of osmication present. Osmication does not saturate with time – increased osmication leads to more osmium deposition onto previously osmicated sites, forming dense polymers. Thus one must test osmication times prior to use by deosmicating a sample and then performing silver intensification. The tissues should remain clear if all osmium has been removed.
Treat osmicated sections on glass slides 10 min with fresh 1% Na metaperiodate in deionized water, rinse 1 min in 0.1 M phosphate buffer (pH 7.4), dip in deionized water & dry. Heavily osmicated tissues may need over 60 min. Perform under a hood and observe all safety precautions for using oxidizing agents.
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