Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulphate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction

Abstract

Four methods based upon the periodic acid-phenylhydrazine-Schiff reaction have been developed for the simultaneous visualization of neutral sugars with periodate oxidizable vicinal diols (hexose, 6-deoxyhexose, N-acetylhexosamine) and either sialic acids or side chain O-acyl sialic acids. In the first of these procedures, the saponification-periodic acid oxidation-2,4-dinitrophenylhydrazine-Azure A-Schiff-saponification (KOH-PA-DNPH-Az-KOH) method, all sialic acids stain Azure blue, neutral sugars with oxidizable vicinal diols stain yellow and mixtures of such components stain in various shades of green. In the second technique, periodic acid oxidation-2,4-dinitrophenylhydrazine-Azure A Schiff-saponification (PA-DNPH-Az-KOH), Azure Blue staining is confined to sialic acids without side chain substituents or which have an O-acyl substituent at position C7, while in the third method, the selective periodate oxidation-borohydride reduction-saponification-periodic acid oxidation-2,4-dinitrophenyl hydrazine-Azure A-Schiff-saponification (PA-Bh-KOH-PA-DNPH-Az-KOH) technique, only sialic acids with O-acyl substituents at positions C7, C8 or C9 (or which have two or three O-acyl side chain substituents) stain Azure blue. Finally in the fourth procedure, periodic acid oxidation-2,4-dinitrophenylhydrazine-Azure A-Schiff-saponification-borohydride reduction-periodic acid oxidation-Schiff (PA-DNPH-Az-KOH-Bh-PAS), sialic acids without side chain substituents or which have O-acyl substituents at C7 stain Azure blue, sialic acids substituted at position C8 or C9 (or which are di- or tri-substituted) stain magenta and neutral sugars stain yellow. Where mixtures of these components are present, a wide range of colours is obtained.