4 6 diamidino 2 phenylindole (dapi)

Cells were fixed in 4% paraformaldehyde at 4 °C for 30 min. After blocking, the cells were incubated with anti-human OC antibody (Bio-Rad) followed by another incubation with secondary antibody conjugated with Alexa fluor 488 (Thermo Fisher Scientific, Waltham, MA, USA). Cell nuclei were also stained with DAPI (Thermo Fisher Scientific). The cells were observed under a fluorescence microscope (BZ-X710; Keyence, Osaka, Japan). OC -positive and negative cells were counted by BZ-II Analyzer software (Keyence, Osaka, Japan) to calculate the percentage of OC -producing cells as follows: %OC producing cells = the number of OC (+) DAPI (+) cells per total number of DAPI (+) cells

The D-17 and A-72 cells (1 × 10⁵ cells/sample) were fixed with 4% paraformaldehyde in PBS for 10 min and quenched with 50 mM NH₄Cl in PBS containing 0.2 mM Ca²⁺ and 2 mM Mg²⁺ (PBSc/m) for 10 min. The cells were treated with blocking buffer (PBSc/m supplemented with 0.5% BSA) for 30 min and incubated with 10 µg/mL of E134Bf or the control blocking buffer for 1 h. The cells were then incubated with Alexa Fluor 488-conjugated anti-dog IgG (1:400; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and DAPI (Thermo Fisher Scientific Inc.) for 45 min. Fluorescence images were obtained using a BZ-X800 digital microscope (Keyence, Osaka, Japan) at a magnification of 40× with a GFP filter channel (green) and a DAPI filter channel (blue). All experiments were conducted in triplicate.

Gastrocnemius muscles were embedded in paraffin and cut into 4 μm sections. After blocking 1 h with 5% bovine serum albumin/PBS containing 0.1% Tween 20 at room temperature, sections were incubated with Alexa Fluor 488 anti-mouse F4/80 antibody (1:100 dilution; 123120, BioLegend, San Diego, CA) and anti-TNF alpha antibody (1:100; ab6671, Abcam, Cambridge, UK) overnight at 4 °C. After three PBS washes, sections were stained 1 h with F(ab')2-Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (1:200; Invitrogen, A-11071, Waltham, MA) at room temperature. DAPI (1:5000; Wako Pure Chemicals Industries, Osaka, Japan) was used for a nuclear stain. The number of F4/80/TNFα double-positive cells and DAPI positive nuclei was determined in five randomly selected 500-μm squares using a fluorescence microscope (BioRevo, Keyence, Osaka, Japan).