Immunohistochemical Analysis of CD4 and IL-21

Paraffin-embedded samples were cut into 5-μm slices, and immunohistochemistry was conducted as previously described (4 (link)). In brief, the mucosa was fixed with paraformaldehyde, followed by antigen retrieval with the EDTA antigen retrieval solution (Servicebio). The processed samples were blocked with 5% bovine serum albumin (BSA) and subsequently incubated with CD4 (Clone RPA-T4, BioLegend) and IL-21 (Abbiotec, Product #253397) Abs, followed by staining of secondary Abs Alexa Fluor^® 488 (Thermo, Product #A-11034) and Alexa Fluor^® 594 (Thermo, Product #A-11005) as well as DAPI (Thermo, Product #A-11034) before the detection. Fluorescence images were captured under a ZEISS IMAGER A1 fluorescence microscope (CARL ZEISS).