Post-embedding staining with high-iron diamine-thiocarbohydrazide-silver proteinate and its application to visualizing sulfated glycoconjugates in cryofixed kidney and cartilage

Abstract

Cryofixation is generally believed to provide optimal tissue preservation. However, certain post-embedding cytochemical reactions, such as high-iron diamine (HID) staining for sulfated glycoconjugates, are not applicable to cryofixed and freeze-substituted tissues. In the present study, the HID technique was therefore adapted for post-embedding staining. HID staining was performed on thin sections of chemically and cryofixed kidney and growth plate cartilage, embedded in Epon and various acrylic-based resins. All resins and most tissue preparation conditions allowed post-embedding staining with HID, albeit to variable degrees. However, no significant cytochemical reaction was obtained with tissue sections of osmicated kidney embedded in Epon. Profile views of re-embedded sections showed that large stain deposits were usually restricted to the surface, whereas small ones were observed throughout the entire thickness of the section. The staining pattern was essentially similar between chemically fixed and cryofixed specimens. In the glomerulus, stain deposits were mainly seen over the free surface of podocyte foot processes and over the lamina rara externa. The pericellular cartilage matrix of chemically fixed specimens often appeared as condensed elements, usually stained with large deposits. In cryofixed tissues this matrix formed a meshwork composed of thin, extended filamentous structures, many of which showed linear arrays of smaller stain deposits. The data presented here indicate that post-embedding HID-TCH-SP staining can be successfully performed on thin sections of tissues embedded in various resins and, as a result, can be further adapted to cryo-prepared specimens to give a high resolution localization of sulfated glycoconjugates in tissues with optimal molecular preservation.