H l alexa fluor 488

5 µm thick paraffin-embedded pellets were deparaffinized, rehydrated, and then treated with protease XXV (AP-9006-005, Thermo Scientific) and hyaluronidase (H6254, Sigma-Aldrich). Sections were then incubated with primary antibody: rabbit anti-collagen I (CL50111AP-1, Cedarlane, ON, Canada), mouse anti-collagen II (II-II6B3, Developmental Studies Hybridoma Bank, IA, USA) using a 1:200 dilution and rabbit anti-collagen X (58632, Abcam, UK) using 1:100 dilution at 4 °C overnight, followed by incubation with a goat anti-rabbit IgG (H&L Alexa Fluor 594, Abcam, UK) with a 1:200 dilution for collagen I, X and goat anti-mouse IgG (H&L Alexa Fluor 488, Abcam) with a 1:200 dilution for collagen II. Sections were then stained with DAPI (4′, 6-diamidino-2-phenylindole, Cedarlane) and mounted with Glycerol and PBS (1:1 ratio). Immunofluorescence was visualized by an Eclipse Ti-S microscope (Nikon Canada, Mississauga, Canada).

For osteogenic and adipogenic protein expression, MSCs were cultured on coverslips with ODM and ADM for 14 days. At day 7, 10, and 14, cells were fixed with 4% paraformaldehyde in PBS, followed by washing with PBS twice. Cells were permeabilized with 0.3% Triton X-100 (Merck KGaA) in PBS, non-specifically blocked with 3% BSA (Thermo Fisher Scientific, Inc.) in PBS, and incubated with mouse anti-RUNX2 antibodies and mouse anti-PPARγ antibodies (1:50; Santa Cruz Biotechnology, CA, United States) at 4°C overnight. After three washes with PBST, the cells were incubated with goat anti-mouse IgG (1:500; H+L; Alexa Fluor 488; Abcam) for 1 h at room temperature and then counterstained with antifade containing DAPI (Invitrogen; Thermo Fisher Scientific, Inc.). The fluorescent images were obtained with a confocal laser scanning microscope and analyzed with FluoView FV1000 Software version 3.01 (Olympus Corp., Tokyo, Japan).