Immunocytochemical Analysis of ECM Proteins

MSCs were washed and fixed with 40 g/L PFA for 30 min at RT. Cells were permeabilized with 0.1% (v/v) Triton X‐100 (Sigma-Aldrich) for 10 min, at RT, rinsed and blocked with 5 g/L BSA/ 0.1% (v/v) Triton X‐100 for 1 h at RT. Then, cells were incubated with primary antibody against collagen type I (COL1, 1:250, Rockland), osteopontin (OPN; 1:250, Santa Cruz Biotechnology), fibrillin-1 (FBN1; 1:100, Thermo Fisher Scientific), collagen Triple Helix Repeat Containing 1 (CTHRC1; 1:50, Santa Cruz Biotechnology) and vitronectin (VTN; 1:25, Santa Cruz Biotechnology) overnight at 4 °C. Cells were washed three times with PBS 1x, 5 min each, incubated with the respective secondary antibody (1:1 000; Thermo Fisher Scientific) for 1 h at RT and washed again. Cell nuclei were stained with 1 μg/mL DAPI (Invitrogen) for 5 min. The antibodies manufacturers and respective dilutions are detailed in Additional file 1: Table SIV. The stainings were visualized under the Leica DMi6000 FFW (Leica Microsystems). Cells incubated with the antibody diluent alone (without primary antibody), followed by incubation with the secondary antibody, were used as negative controls. Semi-quantification of the protein was performed using the ImageJ Fiji software, considering a minimum of 6 distinct fields per condition.