The histochemistry of thiols and disulphides. II. Methodology of differential staining.

The reduction of disulphide bonds by various mercaptans and tri-n-butyl-phosphine (TBP) has been examined in paraffin sections of rat tissues. A "re-reduction' procedure demonstrating any residual disulphides shows that nearly equivalent endpoints are reached by all of the reagents at pH 8.5 and room temperature, though at greatly differing rates. TBP is the reductant of choice in that it acts rapidly, cannot cause the thiolation which is more or less pronounced with certain mercaptans and least reverses the prior alkylation of native thiol groups by iodoacetate or N-substituted malemides. Supporting studies establish that, except in highly compact structures, native as well as generated thiol groups can be visualized with satisfactory completeness and specificity by N-(4-aminophenyl)maleimide followed by a diazotization and coupling sequence. These findings provide the basis for the selective staining of disulphides, either alone or differentiated from native thiols in the same section.