A10680

For immunoblotting, primary antibodies against mCherry (rabbit; Abcam ab167453; 1:1,000), CD9 (rabbit; Abcam ab92726; 1:1,000), β‐actin (rabbit; Abcam ab8227; 1:2000), and TSG101 (mouse, Genetex GTX70255, 1:1,000) were used. The following Odyssey secondary antibodies were used: anti‐mouse, and ‐rabbit antibodies (Li‐COR, LI 926‐68070, LI 925‐32211) at 1:5,000 dilution for the final detection. For immunocytochemistry, Syndecan‐2 (SDC2, rabbit; Santa Cruz sc‐15348; 1:50) was used followed by staining with secondary antibodies conjugated with Alexa 488 (goat anti‐rabbit; Invitrogen A‐10680; 1:500).

The TUNEL assay was performed using the fluorescein-based In Situ Cell Death Detection Kit (Roche; 12156792910) in accordance with the manufacturer’s instructions. Manually dechorionated Tg(fli1a:EGFP) transgenic embryos were fixed in 4% PFA at 4 °C overnight, washed three times with PBST and dehydrated in 100% methanol at −20 °C for more than 2 h. After gradual rehydration, embryos were washed with PBST three times, followed by proteinase K and acetone treatment. After washing 3 times with PBST, the permeabilized embryos were incubated in a mixture containing labeling solution and enzyme solution at a ratio of 9:1 at 4 °C overnight. Finally, the embryos were washed three times with PBST and then were captured by confocal microscopy. Note that anti-GFP (Invitrogen; A-11120) and Alexa Fluor 488-conjugated goat anti-mouse (Invitrogen; A-10680) antibodies were used as the primary and secondary antibodies, respectively.

E16.5 mouse cortical neurons were seeded on 8-well chamber and cultured in vitro for 7 days (DIV7). The neurons were rinsed once with PBS and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature. After washing three times with PBS, cells were permeabilized with 0.2% TritonX-100 in PBS for 10 min. Cells were then washed three times with PBS, blocked with 5% Normal Goat Serum (ThermoFisher) in 1×PBS for RT for 1 h, followed by incubation with primary antibodies at 4 °C overnight. After washing three times with 1×PBS, cells were incubated with corresponding Cy3 conjugated anti-rabbit IgG (A10520, Invitrogen), Alexa Fluor 488 conjugated anti-mouse IgG (A10680, Invitrogen) secondary antibody at RT in darkness for 1 h. After washing 3 × 5 min with 1×PBS, cells were then mounted with DAPI-Fluoromount-G™ Clear Mounting Media (SouthernBiotech, 010020). Fluorescent images were acquired using immunofluorescence microscope.

Immunofluorescence was performed on 10-µm-thick sections. Following permeabilization with PBS containing 0.5% Triton X-100 for 15 min and blocking with 1% BSA in PBS for 60 min at room temperature, the sections were incubated with primary antibodies diluted in PBS containing 1% BSA and 1% Tween 20 at 4 °C overnight. Primary antibodies used in this study include 1:100 CD90 (Abcam #Ab225), 1:100 MYH6/7 (Abcam #Ab50967), 1:100 TNNT2 (Thermo Fisher #MA5–12960), 1:200 ACTN4 (Sigma-Aldrich #A7811), 1:100 GJA1 (Abcam #Ab11370), 1:100 human MYH7 (Abcam #Ab172967), and 1:100 human mitochondria (Abcam #Ab92824). After washing three times with 1% Tween 20 in PBS, the samples were incubated with secondary antibodies for 60 min at room temperature in the dark. Secondary antibodies used in this study include either 1:400 anti-mouse IgG Alexa Fluor 488 (Invitrogen #A10680) or 1:400 anti-rabbit IgG Alexa Fluor 647 (Invitrogen #A21245). After washing again with 1% Tween 20 in PBS, the sections were stained with DAPI solution (VectaShield) for nuclear staining and then mounted on slides. Imaging of heart sections was performed with a Laser Scanning Microscope LSM 880 NLO with Airyscan processing (Zeiss).

For staining intracellular p53 and p21, cells were first labeled with surface markers (Lin⁻Sca1⁺CD48⁻CD150⁺Mac1⁺), fixed, and permeabilized. The cells were then stained with a p53 (9282s, Cell Signaling) or p21 (2947t, Cell Signaling) primary antibody, followed by an Alexa Fluor 488‐conjugated goat anti‐rabbit (A‐10680, Invitrogen) secondary antibody. The cells were then washed twice with PBS and analyzed using flow cytometry.