Antigen retrieval solution

To prepare formalin-fixed and paraffin-embedded (FFPE) tissue sections for immunofluorescence, samples from the duodenum, jejunum, and ileum were fixed in 4% paraformaldehyde and embedded in paraffin according to conventional methods. Briefly, 5-μm thick sections were cut onto gelatinized slides. The slides were deparaffinized and rehydrated and placed in an antigen retrieval solution (SolarBio, China) in a boiling water bath for 30 min. The slides were blocked using goat serum and incubated at room temperature for 20 min. Sections were then stained with mouse anti-chicken IgA-FITC (SouthernBiotech, USA) in humidified chambers overnight at 4°C. After washing with PBS, the nuclei were stained using Hoechst 33,258 (SolarBio, China). The autofluorescence of the FFPE tissue sections were diminished using sodium borohydride according to the method of Davis et al. (2014 (link)). A total of 162 sections (3 groups × 6 birds per group × 3 intestinal segments with 3 replicates) were observed using an inverted microscope (Axio Examiner ZEISS, Germany), and ZEN lite for Windows was used to photograph images under 100× magnification. It should be ensured that more villus and crypt appeared under the microscope; the number of IgA-positive cells of per villus was calculated by Image J software (NIH, USA).