Ultrastructural localization of sulfated complex carbohydrates with a modified iron diamine procedure.

Abstract

A thiocarbohydrazide-silver proteinate (TCH-SP) sequence was applied to thin sections of specimens that had been reacted with the high iron diamine (HID) method for ultrastructural localization of sulfated complex carbohydrates. The exposure to TCH-SP enhanced the electron opacity of HID-reactive sites and increased the sensitivity of the procedure. This held true for HID-reacted specimens whether or not they had been post-treated with osmium tetroxide. However in those not postosmicated after HID, the contrast and specificity appeared superior, as sites of osmiophilia were densified equally in specimens exposed to HID, and unexposed controls, by the final osmium tetroxide-TCH-SP sequence. Staining of immature granules of developing polymorphonuclear neutrophils by HID was intensified by the post-treatment with TCH-SP. In addition, granules of blood mononuclear leukocytes and heterophagosomes of peritoneal macrophages revealed HID affinity and hence content of sulfated mucosubstance that was not evident without the TCH-SP steps. Control procedures which entailed initial exposure of the specimen to FeCl3 or MgCl2 solutions and treatment of thin sections with TCH-SP failed to impart density to these sites.