The histochemistry of thiols and disulphides. IV. Protective fixation by organomercurial-formalin mixtures

Formation of mercaptides as the result of adding organomercuric salts to neutral formalin used for fixation was found to protect protein thiols from autoxidation, provided the tissues were washed in distilled and not tap water. Such bloking, in contrast to that given by HgCl 2 , could be reversed quantitatively by mercaptoethanol made strongly acid to keep it from reducing disulphides. However, some cleavage of disulphides by the mercurials themselves caused slight arbfactual thiol staining in a limited number of sites. Three of the nine compounds tested are sufficiently soluble to penetrate tissues with reasonable speed, stable enough to preclude more than incidental mercurial deposits and currently available commercially. Of them, the diuretic mercurial Mersalyl is at present the protecting, agent of choice since methyl- and ethylmercuric chlorides are too toxic to recommend for routine use.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42846/1/10735_2005_Article_BF01066541.pd

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

The reduction of disulphide bonds by various mercaptans and tri- n -butylphosphine (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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42844/1/10735_2005_Article_BF01003139.pd