Ix73 th4 200 system

Cells were seeded in Matrigel coated 6-well plates overnight to approximately 60% confluence, and washed with cold PBS (HyClone, SH30256.01) twice. The cells were then fixed in 4% paraformaldehyde for 15 min, and incubated in 0.25% Triton X-100 (Amresco, USA, 0694) for 15 min at room temperature. The primary monoclonal antibodies, PAX7 (ABclonal, China, A7335), MYOD1 (ABclonal, A0671), DES (Abcam, UK, ab8976), MYOSIN (Abcam, ab15), and MYOG (Abcam, ab1835) were hatched with the prepared cells at 4 °C overnight. The secondary antibodies, anti-mouse IgG alexa fluor 555 (CST, USA, 4409s) and anti-rabbit IgG alexa fluor 488 (CST, 4412s), were incubated with the cells for 1 h in dark room. Cell nuclei were stained with Hoechst33342 (Sigma, B2261). Images were captured using the OLYMPUS IX73 TH4-200 system (OLYMPUS, Japan).

The cells were cultured in 24-well plates, rinsed twice with phosphate buffer solution (PBS) and fixed with ice-cold Immunostaining Fix Solution (Beyotime Biotechnology, Shanghai, China) for 20 min, followed by washing with PBS two times. Then, the cells were incubated in ice-cold Immunostaining Permeabilization Buffer (Beyotime, China) for 10 min and washed twice with PBS. The cells were then incubated in Immunostaining Blocking Buffer (Beyotime Biotechnology, China) at room temperature for 60 min, followed by washing with PBS three times. Next, the cells were incubated with the following primary antibody, Mouse anti Pig CD163 Monoclonal Antibody (Bio-Rad, Berkeley, CA, USA, MCA2311GA), at 4 °C overnight, and they were washed twice with PBS. Next, the cells were incubated with Goat anti Mouse IgG (H/L) (DyLight^®488, Bio-Rad, USA) for 1 h in the dark at room temperature, following by washing with PBS three times. The cells were then stained with 4′,6-diamidino-2-phenylindole (DAPI) in the dark for 5 min and then rinsed three times with PBS. The images were visualized using an Olympus/IX73 TH4-200 system (Olympus, Tokyo, Japan) or Olympus/FV10i-O system (Olympus, Japan). Three independent experiments were performed for immunofluorescence detection.

C2C12 cells cultured in 12-well plates were washed twice with phosphate-buffered saline (PBS) and fixed with ice-cold 4% paraformaldehyde for 15 min. Then, the cells were again washed twice with PBS and incubated in ice-cold 0.3% Triton X-100 at room temperature for 10 min. The cells were then washed three times and incubated in blocking solution (3% bovine serum albumin, 10% fetal bovine serum and 0.3% Triton X-100) at room temperature for 2 h. Next, the cells were again washed three times and incubated at 4 °C overnight with primary antibodies specific for the following proteins: MyoD (1:100, Santa Cruz, USA, sc-32758×), MyoG (1:100, Abcam, USA, ab1835), and MyHC (1:200, Sigma, USA, M4276). The cells were then washed three times with PBS and incubated with anti-mouse IgG (H + L), F (ab′) 2 Fragment (Alexa Fluor® 555 Conjugate, CST, USA, 4409) for 2 h at room temperature in the dark. Finally, the cells were washed three times with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI) in the dark. Images were acquired using an OLYMPUS IX73 TH4-200 system (OLYMPUS, Japan).