Paraformaldehyde (pfa)

Cells were fixed with 4% paraformaldehyde (Nacalai Tesque). After fixation, the cells were permeabilized with PBS containing 0.2% Triton X-100 (Nacalai Tesque) for 10 min, blocked with 1% BSA fraction V (Millipore Sigma) in PBS, and then reacted with the indicated primary antibodies in PBS for 1 h at room temperature. After washing with PBS three times, cells were incubated with goat anti-mouse IgG Alexa Fluor 647–conjugated (1:1,500 dilution) or anti-rabbit IgG Alexa Fluor 488–conjugated (1:1,000 dilution) secondary antibodies in PBS for 1 h at room temperature. The cells were then counterstained with Hoechest 33342 (Dojindo) (1:2,000 dilution) for 10 min.

The immunostaining and counting of TH+ neurons in zebrafish were performed as previously described^{42 (link)}. Briefly, adult fish were sacrificed by the addition of 0.1% tricaine to the circulating water of the breeding system. Brains were removed, explanted, and fixed with 4% (w/v) paraformaldehyde in PBS (Nacalai) at 4 °C O/N. The specimens were embedded in 2% low-melting agarose, and 200 μm axial sections were prepared using a PRO7 microslicer (Dosaka EM, Kyoto, Japan). Floating slices were incubated with 10 mM sodium citrate buffer, pH 8.5, at 80 °C for 120 min. After washing with PBS containing 1% Triton X-100, sections were blocked with 2% BSA in PBS/1% Triton X-100 buffer for 30 min. These pretreated sections were incubated with a rabbit anti-TH antibody (1/500, AB152, EMD-Millipore, Billerica, MA, USA) O/N at 4 °C. After washing with PBS/1% Triton X-100 buffer, the sections were incubated with anti-rabbit IgG conjugated to Alexa Fluor 594 (1/100, ThermoFisher Scientific) O/N at 4 °C. The sections were washed with PBS/1% Triton X-100 buffer and analyzed using an A1R+ confocal microscope (Nikon).

Histological analysis was performed as described previously [18 (link)]. Briefly, tissues were fixed in 4% paraformaldehyde (Nacalai Tesque), followed by grease removal with 50% (v/v) chloroform (Nacalai Tesque) in ethanol for 2 h, and decalcification in 0.24 M solution of 2NA(EDTA/2Na) and 4NA(EDTA/4Na) (DOJINDO) for 10 days at 4°C. Paraffin sections (5-μm thick) were stained with H&E and analyzed under a microscope (BZ-9000; Keyence).

Cells were fixed with 4% paraformaldehyde (Nacalai Tesque, Kyoto, Japan, http://www.nacalai.co.jp, catalog no. 09154‐14) and permeabilized by 0.5% Triton X‐100 (Acros Organics, Geel, Belgium, http://www.acros.com, catalog no. 215680010) in PBS with Ca²⁺ and Mg²⁺ at room temperature. Blocking was performed by using PBS with 10% serum overnight at 4°C. Cells were incubated with the respective primary antibody diluted in 0.1% serum containing PBS for 1 hour and secondary antibodies in 0.1% serum containing PBS for another hour. Washes were performed twice using 0.1% serum containing PBS. The 4′,6‐diamidino‐2‐phenylindole (1 μg/ml, Thermo Fisher, catalog no. D3571) was used to stain the cell nucleus for 10 minutes. Refer to supplemental online Table 2 for details for primary antibodies used.

To immunostain iPSCMs, cells were fixed with 4% paraformaldehyde (Nacalai Tesque) for 10 min, permeabilized with 0.1% Triton X-100 (Wako) for 5 min, and blocked with PBS containing 5% goat serum (Wako) for 1 hour at room temperature. After the samples were incubated with primary antibodies overnight at 4°C, they were incubated with Alexa Fluor–conjugated secondary antibodies for 1 hour at room temperature. Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific). Images were captured using a confocal microscope (Carl Zeiss, LSM 880) and analyzed using ZEN (Carl Zeiss). The primary antibodies were Lamin A/C (4C11) (1:400; Cell Signaling Technologies, #4777), TNNT2 (13-11) (1:500; Thermo Fisher Scientific, #MA5-12960), and TEAD1 (1:100; Abcam, Cambridge, UK, #ab133533).