A practical technique to postfix Nanogold-immunolabeled specimens with osmium and to embed them in Epon for electron microscopy

Nanogold is a tiny gold probe, freely diffusible in cells and tissues, and is suitable for pre-embedding immunohistochemistry. However, it is necessar y to develop Nano gold to a larger size so that it can be observed by conve ntional transmission electron microscopy. Silver enhancement is usually use d for visualizing Nanogold, but the silver shell produced is unstable in Os O4 and often becomes invisible after OsO4 postfixation, which is necessary for good visualization of ultrastructure. We used silver enhancement with s ilver acetate, followed by gold toning with chloroauric acid, to replace th e silver shell with a more stable gold in order to observe Nanogold after o smium fixation and Epon embedding. This technique is applicable to various intra- and extracellular antigens. For correlative observation of immunolab led specimens by light and electron microscopy, specimens adhered to slideg lasses were embedded in Epon under non-adhesive plastic film. By heating th e Epon sheets after polymerization, these supports were removed without dif ficulty and provided easy correlative observation.