D4v8l

Thymus tissues were fixed with 4% paraformaldehyde and sliced into 10-μm-thick sections. Thymus sections were stained with antibodies specific for Aire (5H12, eBioscience), CCL21 (AAM27, Bio-Rad Laboratories), Ly51 (6C3, BioLegend), and DCLK1 (rabbit polyclonal, abcam) followed by AlexaFluor-conjugated anti-IgG antibodies (Invitrogen). Paraffin sections fixed with 10% formalin solution were deparaffinized and stained with antibody specific for CD3ε (D4V8L, Cell Signaling Technology) followed by AlexaFluor-conjugated anti-IgG antibodies (Invitrogen). Images were visualized with NIS-Elements (Nikon) and analyzed with ImageJ software.

Staining of formalin-fixed paraffin-embedded tumor tissue sections was performed using rabbit–anti-mouse CD3ε antibody (1:150, D4V8L; Cell Signaling Technology, Danvers, Massachusetts, USA), CD4 antibody (1:100, D7D2Z; Cell Signaling) or CD8a antibody (1:400, D4W2Z; Cell Signaling) by the horseradish peroxidase (HRP) method. Antigen retrieval was performed with Citrate Unmasking Solution (Cell Signaling). Before incubation with HRP (Histofine Simple Stain Mouse MAX PO (R), Nichirei Biosciences, Tokyo, Japan), DAB Substrate Kit (Vector Labs, Burlingame, California, USA) was used for visualization of signal. Isotype-matched rabbit IgG was used as a negative staining control. Stained slides were scanned on a DP80 microscope (OLYMPUS, Tokyo, Japan) and digital images were viewed using cellSens (OLYMPUS).