Histochemical Analysis of Stomach Tissue

Whole stomachs with the respective esophagus were fixed and then dehydrated in graded ethanol concentrations (70% to absolute), clarified through two baths in xylene and embedded in paraffin wax at 56–58 °C under a thermostat-vacuum paraffin-embedding bath for 1.5 h. Sagittal sections of 7 μm thick were obtained by using a Reichert–Jung 2050 microtome, then mounted on clean and dry glass slides. Sections were stained with Harris’s hematoxylin-eosin (HE) for morphological evaluation. For histochemical analysis, samples were treated with periodic acid Schiff (PAS), thereby visualizing the vicinal hydroxyls groups; Alcian blue (AB) pH 2.5, thereby visualizing acidic groups pertaining to carboxylated and sulphated complex carbohydrates (sialylated glycoproteins, hyaluronic acid, chondroitin, chondroitin-sulphates A/B/C, heparin, heparansulphate glycosaminoglycan-like material); AB pH 1, thereby visualizing acidic groups pertaining to sulphated complex carbohydrates (chondroitin-sulphates A/B/C, heparin, heparansulphate glycosaminoglycan-like material); and periodic acid Schiff in combination with the Alcian blue (AB/PAS), thereby co-visualizing acidic groups and vicinal hydroxyls [35 ,36 (link),37 ]. Staining solutions were purchased from Bio-Optica Milano SPA: HE (code 05-M06004); AB pH 2.5 (code 05-M26003); AB pH 1 (code 05-M26005); PAS (code 04-130802A); AB/PAS (code 04-163802). All reactions were carried out according to the manufacturer’s instructions.
All stained sections were observed and photographed (ten for 20× for the mosaic and 40× for the particulars) under Leica DM 1000 light microscope. The digital raw images were optimized for image resolution, contrast, evenness of illumination and background using Adobe Photoshop CS5 (Adobe Systems, San Jose, CA, USA).