Alcian blue-periodic acid Schiff (AB-PAS) staining

Specimens from the ileum and proximal colon were collected and stained with AB-PAS for histochemical analysis of GCs as previously described (30). Firstly, 3-4 sections were prepared from each specimen. The sections (4-µm thick) were deparaffinized in xylene at 60°C for 10 min and dehydrated in ethanol solution for 5 min. Oxidation of tissues was performed in 0.5% PA solution at room temperature for 5 min, followed by rinsing in distilled water. Next, sections were stained with Schiff reagent (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 10 min and rinsed in tap water for 5 min. Subsequently, the sections were stained with 1% AB in an aqueous solution of 3% acetic acid (Ph 2.5) at room temperature for 15 min. Finally, they were counterstained with hematoxylin at room temperature for 2 min. All samples were observed under a light microscope at a magnification of ×20.