Light-microscopic detection of acidic glycoconjugates with sensitized diamine procedures

Abstract

To enhance the efficiency and specificity of diamine methods in light microscopy, these methods were sensitized by silver enhancement in combination with trichloro(ethylene) platinate (KTP). The sensitized diamine methods consisted of a diamine (high or low iron diamine: HID or LID), KTP, borohydride reduction (BH) and a physical development (PD) sequence. The new methods have been successfully applied to routinely prepared tissue sections obtained from rat organs, such as salivary glands, stomach, colon, kidney, lung and trachea. In the tissues subjected to the sensitized diamine methods, weakly diamine-stained histological structures exhibited vivid positive reactions. The combined sensitized diamine methods and selective procedures, such as enzyme digestion and chemical modification, have substantiated that these methods were of sufficient efficiency and specificity.

A Histochemical Approach to Correlative Light and Electron Microscopic Detection of Acidic Glycoconjugates by a Sensitized High Iron Diamine Method

“…It is known that sulfated glycoconjugates exhibit positive S-HID reactions in tissues (Hirabayashi 1992) and that heparitinase can specifically degrade heparan sulfates (Hovingh and Linker 1970). In view of these facts and the present results, we suggest that the S-HID reactions of all the basement membranes examined can largely be attributed to heparan sulfate.…”

“…Detailed procedures for the S-HID method have been described elsewhere (Hirabayashi 1992). Briefly, deparaffinized and rehydrated sections were incubated in an HID solution for 60 min at 30C, immersed in 0.5 mM potassium Correspondence to: Y. Hirabayashi,…”

“…However, because suitable methods common to both types of microscopy have not yet been developed for identifying acidic glycoconjugates, only a few attempts to detect such carbohydrates in the same histological site have been reported (Nagato et al 1984;Nagato and Kushida 1985). The sensitized high iron diamine (S-HID) method was developed to detect small amounts of acidic glycoconjugates in tissues by light microscopy (Hirabayashi 1992). Because the final reaction products of the S-HID method are heavy metals, it appears plausible that this method could be adapted for light and electron microscopic histochemistry of acidic glycoconjugates in the same specimen.…”

Histochemical studies of glycosaminoglycans in developing periodontal ligaments of ICR mice

“…Additionally, to detect acidic glycoconjugates in the tissues, a series of the sections were prepared for staining with the sensitized high iron diamine (S-HID) method. Detailed procedures of the S-HID method have been described previously (Hirabayashi, 1992). Deparaffinized and hydrated sections were incubated in an HID solution for 60 min at 30°C, immersed in 0.5 mM potassium trichloro (ethylene) platinum solution for 60 min at room temperature, reduced by 0.2% sodium borohydride solution for 30 sec at room temperature, subjected to a silver enhancement procedure for 8 min at 20°C, and then incubated in appropriate photographic fixer for 2 min at room temperature.…”

Histochemistry of carbohydrates as performed by physical development procedures

“…As an example, HID or LID may be used in combination with potassium trichloro(ethylene) platinum (KTP)-borohydrate (BH) reduction-PD sequence Hirabayashi, 1992). In such combined methods, HID or LID reacted with sulphate or acidic groups of carbohydrates can specifically bind to KTP, which is then reduced by BH to P (platinum), a catalytic metal responsible for subsequent PD procedures.…”