Alexa fluor r 488 goat anti rabbit

For immunofluorescence analysis, after the different treatment, the BMSCs cell line was washed by phosphate‐buffered saline (PBS) three times. Then, cells were fixed in 4% paraformaldehyde for 15 minutes. After the permeabilization and blocking through incubation with 5% FCS, 1% TritonX100 in PBS for 30 minutes at room temperature, the primary antibodies of FoxO1 Mouse mAb (#14952, CST,1:200) were incubated in 5% BSA in PBS overnight at 4°C and followed by Alexa Fluor R 488 Goat Anti–rabbit (ThermoFisher Scientific) at a dilution of 1:400. The slides were mounted by antifade with DAPI. Images were visualized on Olympus microscope using cellSense Dimension software or confocal microscope. To observe the LC3 punches, the cells overexpressing LC3‐GFP protein were fixed in 4% paraformaldehyde for 15 minutes followed by mount of antifade with DAPI. The images were captured by a confocal laser scanning microscope (Leica).

For immunofluorescence, cells were washed by phosphate-buffered saline (PBS) three times. Then, cells were fixed in 4% paraformaldehyde for 15 min and followed by permeabilization and blocking with 7% FCS, 1% TritonX100 in PBS for 30 min at room temperature. Primary antibodies of total β-Catenin (#9562; CST, 1:200) and Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (#8814; CST, 1:800) were incubated overnight in 7% BSA in PBS overnight at 4°C and followed by Alexa Fluor R 488 Goat Anti– rabbit (Thermo Fisher Scientific, United States) at a dilution of 1:400, and finally stained and mounted by mounting medium. Images were visualized on Olympus microscope using cellSense Dimension software.