Alexa fluor 633 goat antimouse secondary antibody

After the end of the experiment, the scaffolds were fixed in 3% paraformaldehyde (Thermo Fisher Scientific) for 15 min and permeabilized in 1% Triton X-100 (Sigma-Aldrich) for 20 min. The actin cytoskeleton and cell nuclei were stained with Alexa Fluor 488 phalloidin (1:1000, A12379, Thermo Fisher Scientific) and DAPI (2 μg/ml, D1306, Invitrogen), respectively. Col-I primary antibody (ab90395, Abcam plc) was added to the medium at a dilution of 1:200 30 min before fixation to facilitate ECM staining, as described previously (44 (link)), and counterstained with AAlexa Fluor 633 goat anti-mouse secondary antibody (A-21052, Thermo Fisher Scientific) at 1:50. YAP was stained with YAP (63.7) primary mouse monoclonal antibody (sc-101199, Santa Cruz Biotechnology) at 1:200 and counterstained with Alexa Fluor 633 goat anti-mouse secondary antibody (A-21052, Thermo Fisher Scientific) at 1:50. α-SMA was stained with primary mouse anti–α-SMA antibody [0.N.5] (ab18147, Abcam plc) at 1:100 and counterstained with Alexa Fluor 546 goat anti-mouse secondary antibody (A-11030, Thermo Fisher Scientific) at 1:100. After washing with phosphate-buffered saline (PBS), primary and secondary antibodies were applied for 30 min each except for Col-I primary antibody, as mentioned above.