Goat anti chicken iga

The duodenum tissues were collected and sectioned in a conventional way (Yu et al., 2015 (link)). First, the sections were removed as paraffin by using xylene and rinsed with PBS 3 times, each time for 5 min. Next, the sections were placed in citrate buffer and bathed at 98°C for 20 min. When cooled to 37°C, rinse 3 times with PBS for 5 min. After 3% H₂O₂–methanol solution and 5% fetal bovine serum were added and incubated at 37°C for 10 min and 30 min, respectively, the sections were incubated with the diluted goat anti-chicken IgA (1:600, Bethyl Laboratories) at 4°C for 12 h. Then 1:1,000 dilution of HRP-conjugated rabbit anti-goat IgG antibodies (Beyotime Biotechnology, Shanghai, China) were added and incubated for 1 h at room temperature before washing. The reaction was terminated with distilled water after the 3,3′- diaminobenzidine (Boster, Wuhan, China) coloration solution was applied for 3 min. Hematoxylin was restained with 40 s, and 1% alcohol hydrochloride was differentiated with 20 s. Finally, sections were sealed by conventional methods. Sections were observed by a light microscope (Nikon Eclipse 80i, Nikon, Tokyo, Japan), and the number of IgA + cells was counted. Ten visual fields were selected from each section for statistical analysis.