Alexa fluor 568 conjugated secondary antibody

Cells were fixed with 4% paraformaldehyde, followed by antigen retrieval (for MRC1) in Tris–EDTA buffer (pH 9.0) or permeabilization (for Nrf2 and CD133) in 0.1% Triton X-100 for 5–10 min. Nonspecific binding sites were blocked with 10% fetal bovine serum (FBS) for 20 min, followed by incubation with an anti-MRC1 (Southern Biotech, Birmingham, USA) used at a dilution of 1:50, anti-chicken CD133 (self-prepared) or anti-Nrf2 (Proteintech, Wuhan, China) at 1:200 overnight at 4 °C. The primary antibody detection was performed with an FITC-labeled or Alexa Fluor 568-conjugated secondary antibody (Abcam, Cambridge, UK). Nuclei were visualized with DAPI (4′-6-diamidino-2-phenylindole) for 5 min. Cells were observed with a fluorescence microscope and photographed. MRC1+ and CD133+ cells in each well were counted in 6 randomly selected high-power fields (× 200).

The cells were fixed by incubation in cold methanol for 2 min and washed three times with phosphate-buffered saline (PBS). They were subsequently incubated with PBS containing 1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 30 min followed by incubation with an anti-MHC monoclonal antibody (Clone MF20, R&D Systems, Minneapolis, MN, USA) at 4°C overnight. The antibody was diluted 1000-fold with PBS containing 1% BSA. The cells were washed and then incubated with Alexa Fluor 568–conjugated secondary antibody (ab175701, abcam, Cambridge, UK) at room temperature for 1 hour. After washing, they were mounted with a medium containing 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Vector Laboratories, Burlingame, CA, USA). Using a fluorescence microscope (IX73, Olympus, Tokyo, Japan), four images were randomly selected from each well. The nuclei were counted using imaging software (cellSens, Olympus). The percentage of nuclei in the myotubes with MHC-positive staining was calculated as the fusion index.

Antibodies against ZO-1 (33-9100) (Thermo Fisher Scientific, Waltham, MA, USA), β-actin (A5316) (Sigma-Aldrich, St. Louis, MO, USA), and GFP (50430-2-AP) (Proteintech, San Diego, CA, USA) were purchased from the indicated sources. The horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences (Amersham, UK). Alexa Fluor 568–conjugated secondary antibody was purchased from Abcam (Cambridge, UK). Nocodazole, 4′,6-diamidino-2-phenylindole (DAPI) and streptavidin beads were purchased from Sigma-Aldrich. Caco-2 cells [American Type Culture Collection (ATCC) number: HTB-37] were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Biological Industries, BioInd., Migdal HaEmek, Israel).