Silver Impregnation for Electron Microscopy

Heavy metal impregnation of specimens for electron microscopy offers a means for differential "staining" of various tissue components and cellular constituents. Of many silver impregnation methods, one of the most useful is Gomori's silver methenamine (1) after periodic acid oxidation (PA-SM) (2, 3). Several authors have applied silver impregnation to electron microscopy (4-7), mainly for the demonstration of connective tissue fibers. Dettmer and Schwarz (3) applied Gomori's silver methenamine to the study of teased preparations. We have adapted this procedure to the impregnation of small blocks of tissue which are then embedded in plastic and sectioned in the usual manner. Procedure Fixation.-Small blocks of tissue (approximately 1 mm. in diameter) were fixed by one or more of the following methods: (a) 4 per cent formaldehyde USP (pH approximately 5.6), 12 to 24 hours at room temperature ; (b) 4 per cent formaldehyde made isotonic by addition of 2 per cent sodium acetate (pH approximately 6.8), 12 to 24 hours at room temperature; (c) 4 per cent formaldehyde in ~/15 phosphate buffer, pH 7.4, 24 to 48 hours in the refrigerator; and (d) formaldehyde-alcohol-acetic add (5 m]. formaldehyde USP 37 to 40 per cent, 5 ml. glacial acetic acid, 90 ml. 70 per cent ethyl alcohol), 2 to 6 hours at room temperature. After fixation tissue was washed in water and stored in 70 per cent alcohol. Fixative (c) affords the best preservation of tissue components. Fixative (d) preserves selectively collagen fibrils and basement membranes at the expense of cell structures. Fixatives (a) and (b) fall in between. Oxidation.-One hour in 0.5 per cent solution of periodic acid in 70 per cent ethyl alcohol at 37°C. followed by several changes of distilled water. Impregnation.-Twenty-four to 48 hours or longer, at 55°C. in a solution of silver methenamine as modified by Jones (8). The solution is changed every 24 hours. At each change one or two blocks of tissue arR removed from the solution, processed through paraffin, sectioned and examined with the light microscope in order to determine the adequacy of impregnation. After im-pregnation is completed, the tissue is washed in several changes of distilled water. It is then postfixed for 30 minutes in a 2 per cent aqueous solution of osmium tetroxide, washed again, and stored in 70 per cent alcohol. Embedding and Sectioning.-The blocks of tissue are dehydrated in alcohols and embedded in butyl meth-acrylate in the usual manner (9). …