Light microscopic histochemical detection of terminal galactose and N-acetylgalactosamine residues in rodent complex carbohydrates using a galactose oxidase--Schiff sequence and peanut lectin--horseradish peroxidase conjugate

Abstract

A technique was investigated for the direct visualization on paraffin sections of galactose and N-acetylgalactosamine residues terminating saccharide chains in complex carbohydrates. Sections were incubated with the enzyme galactose oxidase (GO), which oxidizes the C-6 hydroxyl of galactose or N-acetylgalactosamine (GalNAc) residues, and the resulting aldehyde was visualized by its reaction with Schiff's reagent. Submaxillary and sublingual glands, pancreas, stomach, duodenum, and ileum from mice and rats were stained with the GO-Schiff sequence and results were compared with staining by a peanut lectin-horseradish peroxidase (PL-HRP) conjugate that binds selectively to terminal galactose and preferentially to the terminal dimer beta-D-Gal-(1 leads to 3)-D-GalNAc. Three classes of reactive sites were revealed: 1) those reactive with both GO-Schiff and PL-HRP, 2) those stained with the GO-Schiff sequence but unreactive with PL-HRP, and 3) those GO-Schiff unreactive but PL-HRP positive. Based on the carbohydrate binding specificity of GO and PL, it is suggested that tissue complex carbohydrates in group one contain terminal beta-galactose residues with unmodified hydroxyls at C-2, C-4, and C-6, whereas those in group two contain terminal GalNAc residues. The structure of oligosaccharides in group 3 sites remains enigmatic.