Immunohistochemical Analysis of T-cell Markers

Paraffin-embedded samples were cut into 5-μm sections, and processed for immunohistochemistry as previously described (54 (link)). Briefly, tissues were fixed with 4% paraformaldehyde, followed by membrane permeabilization using 0.2% Triton-X-100. The coverslips were then incubated in 5% BSA, and were sequentially incubated with TCRa7.2 (Clone REA179, Miltenyi) and IL-9 (American Research Products, Product #E-AB-27215) or OX40 (Clone Ber-ACT35, Biolegend) Abs, followed by secondary Alexa Fluor^® 488 Ab (Thermo, Product # A-11034) or Alexa Fluor^® 594 Ab (Thermo, Product # A-11005) and DAPI (Thermo, Product # A-11034) staining before detection. Finally, the coverslips were observed under a ZEISS IMAGER A1 fluorescence microscope (CARL ZEISS) to capture fluorescence images.