Mouse anti cd105

Samples were fixed for 30 min using 4% PFA or 10% formalin for rMSCs cultured on coverslips (n=3) or in microgel scaffolds (n=3), respectively. Samples were washed with PBS 3x for 10 min each. Permeabilization and blocking were performed each for 1 hour at RT in 0.1% TritonX-100 in PBS and 5% bovine serum albumin (BSA) in PBS, respectively. Mouse Anti-N-cadherin (Sigma Aldrich, 10μg/mL), Goat Anti-Mouse FABP4 (R&D systems, 10μg/mL), Rabbit anti-CD73 (Abcam, 1:100), Mouse anti-CD90 (Invitrogen, 1:250), Rabbit anti-CD45, (ThermoFisher, 1:500), and Mouse anti-CD105 (Abcam 1:100) primary antibodies in Cell Staining Buffer (Biolegend) were incubated with rocking overnight at 4°C. The next day, three washes in PBS with 0.05% Tween 20 (PBST) for 10 min each was performed. Then, secondary antibodies (specifically, goat anti-mouse Alexaflour 488 or chicken anti-goat Alexaflour 647 (1:500), DAPI (1:500), Rhodamine Phalloidin (1:300), were incubated for 1 hour at RT. Samples were washed with PBST 3x for 5-10 min. Images were acquired using either the Operetta (Perkin Elmer) for 2D samples or a Nikon Spinning Disc Confocal (40x air objective) or a Laser Scanning Confocal Microscope (20x water objective) for 3D microgel samples.