Ab11369

AMTs were fixed using 4% paraformaldehyde/PBS for 30 minutes and embedded in 13% polyacrylamide gel. 150 μm thick sections were made using a vibratory microtome and stained for cardiac troponin-T (TNNT2, MS-295-P, Thermo Scientific, 1:150 dilution), alpha sarcomeric actinin (α-Actinin, EA53, Sigma, 1:200 dilution), connexin-43 (GJA1, ab11369, Abcam, 1:100 dilution), active caspase-3 (ab2302, Abcam, 1:100 dilution), NKX2-5 (sc-8697, Santa Cruz, 1:50 dilution), and phospho histone H3 (P-histone3, ab32107, Abcam, 1:100 dilution) primary antibodies and Alexa Fluor secondary antibodies. Stained samples were scanned using a confocal microscope and used to generate 3D projection images of the tissue sections in ImageJ. Proliferation and apoptosis were assessed by calculating the percentage of total nuclei positive for phospho histone H3 and active caspase-3, respectively. Myotubes were identified as actinin positive cells containing 5 or more nuclei, since human CMs can contain up to 4 nuclei70 (link).

After cell stretching, the silicone membrane loaded with cardiomyocytes was fixed in 4% paraformaldehyde, cut into tiny pieces and blocked by the donkey serum. Cardiomyocytes were incubated with primary antibodies of MCU (1:50, orb317655, biorbyt), DNM1L/Drp1 (1:100, D6C7, CST) and GJA1/connexin43 (1:200, ab11369, Abcam), respectively. GJA1/connexin43 was used to show the localization and the contour of the cardiomyocyte. Moreover, the contour of the cardiomyocyte indicated by GJA1/connexin43 was used to assess the cellular cross‐sectional area (CSA). After incubation of fluorescent secondary antibodies (Invitrogen Alexa Fluor 488 and 555) and nucleophilic dye 4′,6‐diamidino‐2‐phenylindole (DAPI), the expression of MCU, DNM1L/Drp1 and GJA1/connexin43 in cardiomyocytes inoculated on the silicone membrane was detected by confocal laser scanning microscope (Leica TCS SP8).

Cerebral ischemic cortex or mitochondria were homogenized in lysis buffer. Each sample (50 μg) was loaded onto a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis apparatus. Then, the membranes were blocked with antibodies to Cx43 (Abcam, Cambridge, MA, USA, ab11369, 1:2000), p-Cx43 (Abcam, Cambridge, MA, USA, ab30559, 1:2000), AKT (Abcam, Cambridge, MA, USA, ab25893, 1:500), p-AKT (Abcam, Cambridge, MA, USA, ab81283, 1:500), voltage-dependent anion channel (Abcam, Cambridge, MA, USA, ab34726, VDAC-1; 1:2000), and β-actin (Abcam, Cambridge, MA, USA, ab8227, 1:1000) overnight at 4 °C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Bioss, Beijing, China) in blocking solution for 1 h. Immunoblots were scanned, and protein bands were quantified with Quantitation One software. Relative abundance was obtained by normalizing the density of proteins against that of β-actin or VDAC-1.