TECHNICAL NOTE |
Correspondence to: Yoshifumi Hirabayashi, Dept. of Anatomy, Nagoya City Univ. Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan.
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Summary |
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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. (J Histochem Cytochem 46:767770, 1998)
Key Words: histochemistry, correlative light and electron, microscopy, acidic glycoconjugates, sensitized high iron diamine
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Introduction |
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To 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 (
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Materials and Methods |
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Tissue Preparation for Light Microscopy
After 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 x 7 x 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 µm, mounted on glass slides coated with silane (3-aminopropyl triethoxysilane), and dried in an oven for 56 hr at 60C.
Staining Procedure
A sensitized high iron diamine (S-HID) method was employed. Detailed procedures for the S-HID method have been described elsewhere (
Enzyme Digestion
Before the S-HID staining, some tissue sections were subjected to digestion with heparitinase to identify heparan sulfate. Hydrated sections were incubated in 0.1 U/ml of heparitinase in 0.1 M phosphate buffer (pH 7.0) for 16 hr at 45C (
Tissue Preparation for Electron Microscopy
After light microscopic examination, S-HID-stained sections were exposed by removing the coverslips in QY-1 and wiping up excessive QY-1 surrounding the tissue section. An Epon mixture was dropped on the tissue sections, and eventually Epon 812 mixture-filled beem capsules were placed directly on the exposed sections and polymerized in an oven for 24 hr at 60C. The resin blocks containing the stained sections were removed from the glass slides by heating at 60C. Ultrathin sections were cut at a thickness of approximately 80100 nm on an ultramicrotome (Ultracut E; C. ReicherltJung Optische Werke, Wien, Austria) and mounted on copper grids. The sections, with or without counterstaining with uranyl acetate and lead salts, were examined in a transmission electron microscope (JEM 1200 EX; JEOL Tokyo, Japan).
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Results |
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By light microscopy, a number of histological structures exhibited strong positive reactions with S-HID, including the glomerular basement membrane, mesangial matrices and the surface coat of podocytes and vascular endothelial cells, basement membranes of Bowman's capsules and urinary tubules, and basal infoldings of urinary tubule epithelial cells (Figure 1). Examination of the same sections by electron microscopy revealed satisfactory preservation of histological structures (Figure 2). At lower magnification (Figure 2), the basement membranes of urinary tubules showed positive S-HID reaction. At higher magnifications (Figure 3 and Figure 4), S-HID-reactive histological structures in the glomeruli were recognized to be basement membranes of renal glomeruli and Bowman's capsules, mesangial matrices, certain cytoplasmic granules, and the surface coat of podocytes and vascular endothelial cells.
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When visualized by light microscopy, heparitinase digestion presented a variable decrease in S-HID staining intensity in the basement membranes of renal glomeruli, Bowman's capsule, and urinary tubules (Figure 5). However, positive S-HID reactions in the glomerular mesangial matrices were decreased only slightly in intensity (Figure 5). At the ultrastructural level (Figure 6 and Figure 7), the S-HID reaction in the basement membranes of the same renal glomeruli was either abolished or markedly diminished in intensity. However, large amounts of S-HID reaction product persisted in the cytoplasm and surface coat of podocytes and vascular endothelial cells after digestion with heparitinase (Figure 6 and Figure 7). In control tissue sections for the digestion with heparitinase, images obtained by the S-HID staining were almost identical to those in intact sections (not shown).
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Discussion |
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According to two previous studies (
In the present study, the positive S-HID reactions of the mesangial matrices, the surface coat of the podocytes, and vascular endothelial cells were not affected by the digestion with heparitinase. According to previous reports (
This newly developed and relatively simple approach allows precise correlation of light and electron microscopic S-HID reaction products. Such an approach could contribute generally to a precise analysis of acidic glycoconjugates in light and electron microscopic histo- and cytochemistry.
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Literature Cited |
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