Yap1 sc 101199

Cells were fixed in 4% formaldehyde/0.2% Triton-X (Sigma, Dorset, UK), and after blocking (3% (w/v) in BSA), it was incubated with primary and secondary antibodies. The following antibodies were used for immunofluorescence: COLTypeI, ab88147 from Abcam; FN1, F3648 from Sigma; YAP1 sc-101199 from Santa Cruz Biotechnology; Alexa Fluor™ 594 Phalloidin, A12381, Alexa Fluor™ 488 anti-mouse, A11001 and Alexa Fluor™ 594 anti-rabbit, A-11037, from Thermo Fisher.

Following culture, LMS were washed in PBS and then fixed in 4% formaldehyde solution for 15 min at room temperature. LMS were permeabilized in 1% Triton X‐100 and blocked in 10% foetal bovine serum, 5% bovine serum albumin and 10% horse serum in PBS solution for 3 h at room temperature. LMS were incubated with the corresponding primary antibody αSMA (M0851 mouse monoclonal antibody, Dako, 1:1000), Collagen I (ab34710 rabbit polyclonal antibody, Abcam, 1:1000), Vimentin (pa1‐10003 chicken, Invitrogen, 1:4000), Cardiac troponin (ma5‐12960, Thermofisher, 1:1000), YAP1 (sc‐101199, Santa Cruz, 1:100) in PBS at 4°C overnight, and then washed three times for 30 min. LMS were incubated in the secondary antibody in PBS for 2 h at room temperature. LMS were then washed three times for 30 min, stained with Hoechst (Thermofisher, 1:1000) during 15 min, washed again three times for 15 min and stored in PBS at 4°C. Immunolabelled slices were imaged using a confocal microscope (Zeiss LSM‐870).

Antigen retrieval and 3,3'-diaminobenzidine staining was performed as previously described [49 (link)]. PicroSirius Red Stain Kit was used to stain for collagen. Antibodies used in immunohistochemistry were as follows: ACTA2/α SMA, 180106 from Invitrogen; COL1, ab150681 from Abcam; FN1, F3648 from Sigma; YAP1, sc-101199 from Santa Cruz.