Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and itsO-acyl variants orO-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.

Glycoproteins in the Buccal Epithelium of a Carp, Cirrhinus mrigala (Pisces, Cyprinidae): A Histochemical Profile

“…The sections were then subjected to saponification as described in method 27 and stained with AB1.0/PAS (method 19). PA/Bh/KOH/AB2.5/PAS (periodic acid–saponification–sodium borohydride–alcian blue pH 2.5/periodic acid Schiff's reaction): The method was similar to the method 28 but the sections were stained with AB1.0/PAS (method 19) in place of AB2.5/PAS (method 16). PA/Bh/KOH/PAS (periodic acid–sodium borohydride–saponification–periodic acid Schiff's reaction): The sections treated with PA/Bh/KOH as described in the method 28 were stained with PAS (method 2). PA/T/KOH/PAS: (periodic acid/thionin Schiff–saponification/periodic acid Schiff's reaction): The sections oxidized with 0.5% aqueous periodic acid for 1 h were treated with thionin Schiff's reagent (Van Duijn, ) for 1 h, washed in running tap water for 10–15 min, rinsed in distilled water and transferred to 70% ethyl alcohol. The sections were subjected to saponification and then stained with PAS (method 2). PA/T/KOH/Bh/PAS (periodic acid/thionin Schiff–saponification–borohydride/periodic acid Schiff's reaction): The sections treated with PA/T/KOH, as described in method 31, were subjected to borohydride reduction as per description in the method 28 and then stained with PAS (method 2). KOH/PA/DNPH/Az/KOH: (saponification–periodic acid–2,4‐dinitrophenylhydrazine/azure A Schiff–saponification reaction): The sections, following saponification and periodic acid oxidation, described in the methods 27 and 28, respectively, were treated with a saturated solution of 2,4‐dinitrophenylhydrazine in 1M HCl for 2 h, washed in running tap water for 10–15 min and were kept in distilled water for 16–17 h. The sections were stained with azure A Schiff's reagent (prepared by the method of Park et al., ) for 6 h, washed in running tap water for 10 min and then were subjected again to saponification. PA/DNPH/Az/KOH (periodic acid–2,4‐dinitropheny‐lhydrazine/azure A Schiff–saponification reaction): The method was similar to the method 33 but sections were not subjected to prior saponification. PA/DNPH/Az/KOH/Bh/PAS (periodic acid–2,4‐dini‐trophenylhydrazine/azure A Schiff–saponification–borohydride/periodic acid Schiff's reaction): The sections treated with PA/DNPH/Az/KOH (method 34) were subjected to borohydride reduction, described in method 28 and then stained with PAS (method 2). PA/P/T/KOH/Bh/PAS (periodic acid–phenylhydrazine–thionin schiff–saponification–borohydride/periodic acid Schiff's reaction): The method was similar to the PA/T/KOH/Bh/PAS (method 32), but after periodic acid oxidation, the sections were treated with phenylhydrazine, described in the method 25. PA/Bh/KOH/PA*/Bh/PAS (periodic acid–borohydride–saponification–selective periodate oxidation–borohydride/periodic acid Schiff's reaction): The sections treated with PA/Bh/KOH sequence, described in the method 28, were cooled to 4°C, oxidized with pre‐cooled freshly prepared 0.4 mM periodic acid in 1M HCl at 4°C for 1 h and washed in running tap water for 10–15 min. The sections were then subjected to borohydride reduction, described in method 28, and stained with PAS (method 2).…”