IHC analysis was conducted as previously described (Lee et al. 2018 (link)). Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min in order to remove endogenous peroxidase. Antigen retrieval was carried out with sodium citrate buffer (0.1 M) using the microwave method. Next, the slides were incubated with normal serum so as to block non-specific binding, then incubated for 1 h with primary antibodies (diluted 1:100 to 1:200) such as TNF-α (MyBioSource, San Diego, CA), IFN-γ (sc-74104, Santa Cruz Biotechnology, Dallas, TX), TGF-β (MBS462142, MyBioSource, San Diego, CA), CXCL1 (PAB8798, Abnova, Taipei, Taiwan), CCL-2 (PAB16617, Abnova, Taipei, Taiwan) and CCR2 (Biorbyt, orb137093, Cambridge, UK). The slides were incubated for 10 min with biotinylated secondary antibodies (PK-7800, Vector Laboratories, Burlingame, CA) and horseradish peroxidase-conjugated streptavidin. Finally, signals were detected using the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution, and the cells were counterstained with Mayer’s haematoxylin.