Immunohistochemical Analysis of CD4 and CD103

Paraffin-embedded samples were cut into 5 μm sections, and processed for immunohistochemistry as previously described (Wu et al., 2018b (link)). Briefly, tissue sections were fixed with 4% paraformaldehyde, followed by membrane permeabilization using 0.2% Triton-X-100. Then, the coverslips were incubated in 5% BSA, and were sequentially incubated with primary CD4 (Clone T4, Biolegend) or CD103 (Ber-ACT8, Biolegend) antibody and secondary Alexa Fluor® 488 secondary Ab (Thermo, Product # A-11034) or Alexa Fluor® 594 secondary Ab (Thermo, Product # A-11005) before mounting. Finally, the coverslips were observed under a ZEISS IMAGER A1 fluorescence microscope (CARL ZEISS) to capture fluorescence images.