USE OF A WATER SOLUBLE CARBODIIMIDE AS A FIXING REAGENT

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Abstract
An attempt was made to apply water soluble carbodiimide (WSC) to enzyme histochemistry as a fixative. WSC is a bifunctional reagent which reacts with carboxyl and amino groups of proteins at neutral pH, forming, intermolecular cross-links. The proportion of cross-linked protein in the tissue fixed with the optimal concentration of WSC (40 mg/ml in either 0.1 M phosphate or cacodylate buffer, pH 7.0-7.4) was about 90%. This percentage is almost the same as that obtained by use of 2% glutaraldehyde. Examination with the EM of [mouse] liver, kidney, intestine, pancreas and muscle fixed with WSC at the concentration of 40 mg/ml in 0.1 M phosphate buffer (pH 7.4) and postfixed with 1% OsO4 revealed fine structure which was preserved as well as if glutaraldehyde had been used as the fixative. Enzyme assay, carried out on some enzymes to check the degree of decrease in enzymatic activity after the treatment with WSC, showed that the grade of decrease varied from one enzyme to another. When WSC was used as a fixative fine structure was well preserved. In the case of most of the enzymes examined a higher rate of enzyme activity remained in the insoluble fraction of the tissue homogenate that had been treated with WSC than in glutaraldehyde fixation. WSC promises to be a useful fixative for use in enzyme histochemistry.