F4 80

Samples were fixed for 20 h in Mildform 10N (Wako), embedded in paraffin, and then subjected to hematoxylin and eosin staining (Meyer’s hematoxylin solution and 1% eosin, Muto Pure Chemicals Co., Ltd., Tokyo, Japan) as well as immunohistochemistry using primary antibodies against F4/80 (Cedarlane Laboratories, Ontario, Canada). Oil Red O staining (Nacalai Tesque) was performed on frozen sections. For the image analysis, a microscope BZ-X710 (Keyence, Osaka, Japan), BZ-X Viewer version 1.3.1.1 (Keyence), and BZ-X Analyzer version 1.3.1.1 (Keyence) were used.

Tissues were formalin-fixed and paraffin-embedded. Neutrophil (CD11b⁺Ly6G⁺ cells) were stained as described,^{13 ,18} (link) and counted in 3 random fields per section.
For Mac2, F4-80, and Ki67 staining, after blocking with 3% of H₂O₂ for 20 min at room temperature (RT), and horse serum 1:75 in PBS, 20 min RT; sections were incubated overnight at 4°C with anti-MAC2 (Cedarlane, Burlington, Ontario, Canada), -F4-80 (Biorad, Hercules, CA, United States) or -Ki67 (Cell Signaling, Danvers, MA, United States) in PBS. Tissue sections were then washed and incubated with IgG biotinylated antibody for 30 min at RT, washed in PBS and incubated in ABC reagent (Vector Laboratories, Burlingame, CA, United States) for 60 min at RT, PBS washed, incubated with 3,3′-diaminobenzidine as substrate (Sigma); and haematoxylin counterstained. Positive cells were counted in 3 fields per section. Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) was performed according to the manufacturer (TMR Red, Roche, Basel, Switzerland).