There are various methods for staining mucus substances, such as AB-PAS staining, mucus HID-AB staining, standard Alcian blue staining, etc. Most of the above methods utilize the principle that Alcian blue belongs to cationic dyes that can bind to acidic groups, i.e., the principle that Alcian blue forms insoluble complexes with anionic groups such as carboxylate and sulfate contained in tissues.

Mucin staining solution (mild methylation method) belongs to one kind of chemical modification and blocking method, which is based on the principle of using an alkaline solution containing alcohol to cleave the O-acetyl group in enzyme-resistant sialic acid, and the removal of the O-acetyl group and the formation of hydroxyl groups in the side chain of sialic acid restores the reactivity of sialic acid to PAS; the carboxyl group within the tissues ionized at a pH of 2.5 in the Alcian staining solution carries a negative charge, which binds to the ali The cations in New Blue form a salt bond that stains tissues with carboxyl groups (e.g., proteoglycans/hyaluronic acid as well as epithelial acidic mucins), and is mainly used to identify acidic groups in mucins.