Immunofluorescence staining was performed as described previously.22 (link)
Briefly, MLOY4 cells were cultured in 35 mm confocal dishes at a density of 2 × 105 cells/dish for 24 hours. They were then fixed in 4% paraformaldehyde for 15 minutes, rinsed with PBS three times, permeabilized with 0.5% Triton X-100 (Beyotime) for 15 minutes, and blocked with 5% goat serum for one hour. The samples were then incubated with the anti-Cx43 rabbit monoclonal antibody (1:1,000, Abcam, UK) overnight at 4°C. The secondary antibody was fluorescein isothiocyanate (FITC) fluorescent antibody (1:1,000, Abcam, UK) and TRITC-conjugated phalloidine (1:200, Yeasen Biotechnology), which was used to label the cytoskeleton. Nuclei were counterstained with DAPI (Abcam, UK).
The optimal cutting temperature (OCT) compound embedded femur was sliced into 10 μm-thick transverse sections. The slices were incubated with primary antibodies against voltage-dependent anion channels (VDAC) (1:1,000, Abcam, UK) at 4°C overnight. The secondary antibody was CY5 fluorescent antibody (1:1,000, Abcam, UK), and the slides were sealed by mounting medium with DAPI. Images were captured with a contained confocal laser scanning microscope system (Olympus).
Briefly, MLOY4 cells were cultured in 35 mm confocal dishes at a density of 2 × 105 cells/dish for 24 hours. They were then fixed in 4% paraformaldehyde for 15 minutes, rinsed with PBS three times, permeabilized with 0.5% Triton X-100 (Beyotime) for 15 minutes, and blocked with 5% goat serum for one hour. The samples were then incubated with the anti-Cx43 rabbit monoclonal antibody (1:1,000, Abcam, UK) overnight at 4°C. The secondary antibody was fluorescein isothiocyanate (FITC) fluorescent antibody (1:1,000, Abcam, UK) and TRITC-conjugated phalloidine (1:200, Yeasen Biotechnology), which was used to label the cytoskeleton. Nuclei were counterstained with DAPI (Abcam, UK).
The optimal cutting temperature (OCT) compound embedded femur was sliced into 10 μm-thick transverse sections. The slices were incubated with primary antibodies against voltage-dependent anion channels (VDAC) (1:1,000, Abcam, UK) at 4°C overnight. The secondary antibody was CY5 fluorescent antibody (1:1,000, Abcam, UK), and the slides were sealed by mounting medium with DAPI. Images were captured with a contained confocal laser scanning microscope system (Olympus).
