Anti mouse alexa fluor 488

All chemicals were purchased from Sigma (Zwijndrecht, the Netherlands) unless indicated otherwise. The polyclonal antibody against 3β hydroxysteroid dehydrogenase (HSD3B) was a kind gift from the late Dr. Payne (Stanford, CA, USA). Biotinylated horse-anti-mouse antibody, alkaline phosphatase goat-anti-rabbit antibody, and Vectastain ABC-kit Elite were purchased from Vector Laboratories (Burlingame, CA, USA). Mouse-anti-bromodeoxyuridine (BrdU) and sheep-anti-BrdU antibodies were obtained from Beckton and Dickinson (lot nr 10100, Mountain View, CA, USA) and Abcam (ab1893, Cambridge, England), respectively. Polyclonal anti-SOX9 rabbit antibody was purchased from Millipore (lot nr 2065999, Temecula, CA, USA), whereas the secondary fluorescent antibodies anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 were obtained from Abcam; DAPI was obtained from Sigma (D9564, St. Louis, MO, USA). Acetylated BSA (BSA-c) was purchased from Aurion (Wageningen, the Netherlands). The radio-immuno assay (RIA) kits for determination of T₃, T₄, and testosterone were obtained from DSL (Webster, TX, USA). SACcel, the secondary donkey-anti-rabbit antibody complex used in the in-house RIA analyses, was obtained from Welcome Reagents (Beckenham, UK.).

For immunostaining, hiPSC-CMs were plated on thin black bottom 96 well plates (Corning). Cells were washed using PBS followed by fixing using 5% PFA (Sigma-Aldrich) for 15 min followed by 2x PBS washes. Fixed cells were then washed for 15 min in 0.1% Triton X-100 (Sigma-Aldrich, Gillingham, UK) in PBS followed by 3x further washes in PBS. Fixed cells were then blocked in 4% Goat Serum (Sigma-Aldrich, Gillingham, UK) in PBS for 1 h followed by a further 5 min PBS wash. Primary antibody was added to the plates, diluted in 4% Goat Serum in PBS (Alpha Actinin 1:800, Abcam) and left overnight at 4 °C on a rocking plate. The following day fixed cells were washed with 0.1% Tween (Sigma-Aldrich, Gillingham, UK) in PBS (3 × 10 min) followed by secondary antibody staining [Anti-Mouse Alexa Fluor^® 488 (Abcam, Cambridge, UK) diluted 1:500 in 4% Goat Serum (Sigma-Aldrich, Gillingham, UK) in PBS] for 1 h at room temperature. The fixed cells were then washed 3 times with 0.1% Tween-PBS (Sigma-Aldrich, Gillingham, UK) followed by incubation in PBS supplemented with DAPI (Tocris, Abingdon, UK) at 1:500 for 10 min. A further wash was performed using PBS after which the fixed cells were submerged in PBS and the plate wrapped in foil prior to imaging using the EVOS M5000 imaging system.