DIAMINE METHODS FOR DIFFERENTIATING MUCOSUBSTANCES HISTOCHEMICALLY

Abstract

A mixture of N,N-dimethyl- m-phenylenediamine (HCI)2 with the para isomer at pH 3.5-4.0 stains acid mucosubstances selectively. Various sialomucins as well as sulfomucins differ in their reactivity with this mixed diamine reagent and in the manner in which the pH and ionic strength of the solution affect their staining. Prior periodate oxidation eliminates or markedly decreases the staining of some sulfo- and sialomucins but has no influence on the reactivity of others. Both sulfated mucosaccharides and sialomucins differ also according to whether or not their affinity for azure A, colloidal iron or alcian blue persists following periodate oxidation and exposure to the meta diamine. Azurophilia of nuclei at pH 4.0 is abolished by meta diamine used after Feulgen hydrolysis. Ferric chloride added to a solution of both diamines allows selective demonstration of most acid mucosubstances. This "low iron" diamine method followed by alcian blue stains sulfomucins and many sialomucins black, but other sialomucins blue. Some epithelia contain a mixture of blue and black-stained sialomucins. A variant technique uses a higher concentration of iron and diamines. This colors most sulfomucins black while leaving nonsulfated acid mucosubstances unstained. This high iron diamine method followed by alcian blue stains sulfomucins black and sialomucins blue. Results with the "high iron" diamine-alcian blue sequence indicate that sulfomucin occurs in some sites and sialomucin in others; in many epithelia a mixture of the two mucosubstances coexists either in the same on in different cells. Similar differentiation was obtainable also in many but not all sites with either the combination aldehyde fuchsin-alcian blue procedure on by assessment of differences between the results of alcian blue pH 2.5-PAS and alcian blue pH 1.0-PAS sequences. Applied to the surface mucous layer of certain intestinal and genitourinary epithelia, the methods differentiate areas of sulfomucin content alternating with others of sialomucin content. This is believed to indicate biogenesis of this secretion in the Golgi zone of nongoblet epithelial cells since staining of the surface mucous layer parallels that in the Golgi zone and often not that of neighboring goblet cells.