New cytochemical method for adenylate cyclase and guanylate cyclase with dimethyl sulfoxide.

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Abstract
To assess the appropriateness of the lead nitrate method using dimethyl sulfoxide (DMSO) for the cytochemical demonstration of adenylate cyclase (ACLase) and guanylate cyclase (GCLase), a biochemical assay of ACLase and GCLase was carried out in parallel with the cytochemical study. Rats tissues were fixed in a mixture of 2% paraformaldehyde, 0.25% glutaraldehyde and 5% DMSO in 0.1 M cacodylate buffer, pH 7.4, for 30 min, and then washed with 5% DMSO in 0.1 M cacodylate buffer. Vibratome or microslicer sections were incubated in the following medium for 15-60 min at 37.degree. C. The medium for ACLase contained 80 mM Tris-maleate buffer, pH 7.4; 4 mM MgSO4; 10 mM NaF; 2 mM theophilline, 2 mM lead nitrate; 0.25 M sucrose; 5% DMSO; 2.5 mM levamisole; 0.5 mM AMP-PNP [adenyl imidodiphosphate]. Medium for GCLase: 80 mM Tris maleate buffer, pH 7.4; 3 mM MnCl2 or MgSO4; 2 mM theophylline; 2 mM lead nitrate; 0.25 M sucrose; 5% DMSO; 2.5 mM levamisole; 0.5 mM GMP-PNP. Some reaction medium for GCLase contained NaN3. ACLase and GCLase activites were biochemically and cytochemically enhanced by adding DMSO to the incubation medium as well as to fixatives. Cyclases were localized in the plasma membranes, gap junctions, endoplasmic reticulum and Golgi apparatus.