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

Abstract

SUMMARY A sensitized high iron diamine method is among the reliable and useful histochemical means of detecting acidic glycoconjugates by light microscopy. Because the final reaction products obtained using this method are heavy metals, it can be applied to specimens for visualization by both light and electron microscopy. In this study the high iron diamine method was utilized successfully as a correlative light and electron microscopic method for detection of acidic glycoconjugates. T o fully elucidate the structural and functional details of cells and tissues, it is often necessary to extend results obtained by light microscopy to the ultrastructural level. Because the procedures for tissue preparation and staining are usually quite different, it is rather difficult to examine the same specimen by both light and electron microscopy. Since 1981, several methods have been developed to correlate light and electron microscopic examinations histologically and histochemically (Kushida and Kushida 1981;Nagato et al. 1984;Nagato and Kushida 1985; Bowser 1983, 1985;Kushida et al. 1993). 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. Here we report the development of a correlated light and electron microscopic approach using the S-HID method for detecting acidic glycoconjugates in the same tissues. Materials and Methods Tissue Preparation for Light MicroscopyAfter ether anesthesia, six male Wistar rats were perfused via the left ventricle with a physiological saline solution containing 2.5 mg/dl heparin, followed by perfusion with 4.0% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) containing 7.5% sucrose (fixative solution) for 10 min at room temperature (RT). Kidneys were dissected out, cut into tiny pieces (7 ϫ 7 ϫ 3 mm), and immersed in the same fixative solution overnight (for approximately 16 hr) at 4C. After rinsing with 0.1 M phosphate buffer (pH 7.4) containing 7.5% sucrose, the tissues were dehydrated in an ethanol series of ascending concentrations, immersed in methyl benzoate, cleared in xylene, and embedded in paraffin. Sections were cut at a thickness of approximately 4 m, mounted on glass slides coated with silane (3-aminopropyl triethoxysilane), and dried in an oven for 5-6 hr at 60C. Staining ProcedureA sensitized high iron diamine (S-HID) method was employed. Detailed procedures for the S-HID method have been described elsewhere (Hirabayashi 1992). Briefly, depara...

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