TISSUE FIXATION WITH DIIMIDOESTERS AS AN ALTERNATIVE TO ALDEHYDES I. COMPARISON OF CROSS-LINKING AND ULTRASTRUCTURE OBTAINED WITH DIMETHYLSUBERIMIDATE AND GLUTARALDEHYDE

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Abstract
Diimidoesters are bifunctional reagents which react with the ε-amino group of lysine, forming intermolecular cross-links with minimal alteration of the native properties of proteins. To determine the optimal conditions for tissue fixation with diimidoesters, a cross-linking assay was developed based on the assumption that protein aggregates formed by intermolecular cross-links would be water-insoluble. With this assay method, the proportion of insoluble protein in fresh liver was 16%, while optimal conditions for cross-linking with the diimidoester, dimethylsuberimidate (DMS) (16-20 mg/ml, 0.02 M Ca++ and 0.15 M Tris-HCl, pH 9.5), rendered 92.1% insoluble and permitted only 3.3% of the protein to diffuse into the fixative solution. The smaller diimidoesters, dimethyladipimate and dimethylmalonimidate, rendered 74 and 16% of the protein insoluble, respectively. Fixation with various aldehydes produced insoluble fractions varying from 74% to over 90%. Ultrastructurally, hepatocytes of DMS-fixed liver and glutaraldehyde-fixed liver were similar except that with the former fixative the Golgi saccules and smooth endoplasmic reticulum were dilated and mitochondrial matrices exhibited an increased electron density. Furthermore, the appearance of glutaraldehyde- and DMS-fixed liver was more readily correlated with the degree of cross-linking than with the pH of the fixative solution per se. The results of this study indicate that DMS is a potentially useful fixative for light and electron microscopy.

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