Pa1 26204

Primary antibodies used in this study were: recombinant rabbit anti-cellular communication network factor 2 (CCN2) monoclonal antibody diluted to a concentration of 1:1,000 (Abcam, ab209780); rabbit anti-bone morphogenetic protein 1 (BMP1) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; PA5-103660; RRID: AB_2852994); rabbit anti-collagen type I (COL1) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; PA1-26204; RRID: AB_2260734); rabbit anti-suppressors of mothers against decapentaplegic homolog 2 (SMAD2) polyclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; 51-1300; RRID: AB_2533896); rabbit anti-phosphorylated SMAD2 (pSMAD2) monoclonal antibody diluted to a concentration of 1:1,000 (Thermo Fisher Scientific; MA5-15122; RRID: AB_10978317); and mouse anti-beta actin (β-actin) monoclonal antibody diluted to a concentration of 1:5,000 (Thermo Fisher Scientific; MA1-91399; RRID: AB_2273656).
For near-infrared fluorescence detection, IRDye^® 800CW donkey anti-rabbit and IRDye^® 680RD donkey anti-mouse secondary antibodies (LI-COR, 926-32213 and 926-68072, respectively) were used.

The ECM of the decellularized samples was stained for visualization of laminin, fibronectin, or collagen I separately. For ECM staining, the fixed samples were incubated in blocking buffer (1% bovine serum albumin and 1% Tween 20 in PBS) at room temperature for 1 h. Then, the cultures were incubated with anti-fibronectin polyclonal primary antibody (ab2413, 1:200) (Abcam, Cambridge, United Kingdom), anti-laminin polyclonal primary antibody (PA5-16287, 1:250) (ThermoFisher Scientific, Carlsbad, CA, USA), or anti-collagen I polyclonal primary antibody (PA1-26204, 1:200) (ThermoFisher Scientific) for 2 h at room temperature. The cells were washed 3 times with PBS and incubated 2 h at room temperature with the secondary antibody Alexa Flour 488 anti-rabbit (A11034, 1:800) (ThermoFisher Scientific). Cultures with cells were labelled for detection of fibronectin as described above and then the cytoskeleton was stained with Alexa Flour 594 phalloidin (A12381, 1:100) (ThermoFisher Scientific). Then, the cultures were washed three times with PBS and the cell nuclei were stained with 4′,6-diamidino-2-phenylindole DAPI (1 µg/ml) (ThermoFisher Scientific) for 2 min at room temperature. All samples were stored in PBS protected from light before microscopy.