Immunohistochemical Analysis of Intestinal IgA+ Cells

IgA⁺ cells were identified with an immunohistochemical staining method described previously [15 (link)]. Briefly, fixed intestinal samples were embedded in paraffin, sectioned at a thickness of 6 µm, and mounted onto glass slides. Endogenous peroxidase activity was blocked by incubation with 3% H₂O₂ in methanol for 20 min. After rinsing three times with PBS, the slides were successively incubated with a goat anti-mouse IgA antibody (1:400) and HRP-conjugated rabbit anti-goat IgG antibody (1:250). 3, 3′-Diaminobenzidine (DAB) (BOSTER, Wuhan, China) detection was performed according to the manufacturer’s instructions. Images were captured with a DS-U3 camera interface (Nikon, Tokyo, Japan) and evaluated using the image analysis software Image-Pro Plus 6.0. Three fields per slide were randomly selected at 400× magnification, and the integrated optical density (IOD) was analyzed.
After hematoxylin–eosin staining, the number of iIELs in five different fields of intestinal villi on each slide was counted to statistically analyze the data.