Histochemical studies of the formazan reaction: I. A theoretical review of some of the factors that can affect the conversion of periodate‐oxidized mucosubstances into formazans

SYNOPSIS At present, the chemical structures of macromolecules actually in tissues cannot be determined, mainly because histochemical techniques are rarely specific for a particular chemical group. This field might prosper if it were possible to make use of the unique ability of aldehydes to be converted into highly‐coloured formazans. This is achieved in vitro by condensing aldehydes with an arylhydrazine and coupling the hydrazone thus formed with a diazotized aromatic amine at a pH around 9. However, in situ this may be more complicated. For example, when periodate‐oxidized mucosubstances (containing engendered aldehydes) are converted into formazans by a similar sequence, coloured tetrazenes and azo‐derivatives, among other products, may be formed as well. Some of the many factors which can affect the conversion of periodate‐oxidized mucosubstances into formazans are reviewed in this paper. They include the nature of the “aldehydes” engendered in mucosubstances by periodic acid; the stabilities of periodate‐oxidized mucosubstance arylhydrazones; the course of the coupling reaction between diazon‐ium salts and mucosubstance arylhydrazones; and the presence of groups other than ofc‐glycols in the mucosubstance macromolecule (e.g. sulphate, carboxyl).