Tunel staining

Cells (1 × 10⁴) were cultured for overnight and exposed to the indicated drugs for 24 hours. Then, measured the caspase-3 activity by caspase-Glo 3 kits (Sigma) and cell apoptotic ratio by Annexin V Apoptosis Detection Kits (BioVision). The apoptotic cells were detected by TUNEL staining (Takara).

Pregnant dams received an intraperitoneal injection of BrdU, 50 mg/kg body weight. For cell cycle exit assays, pregnant females carrying E13.5 or E14.5 embryos were injected with BrdU and the embryos were collected at E14.5 and E15.5, respectively, and processed for immunohistochemistry. For birthdating assays, pregnant females carrying E11.5, E13.5, E15.5, or E17.5 embryos were injected with BrdU and embryos were collected at P0. Sections were denatured in 1.5 N HCl for 30 min at 37°C, followed by neutralization in 0.1 M sodium borate (pH 8.5) before incubation with the primary antibodies. The cortical thickness was divided into 10 parallel subregions (bins) and the number of BrdU+ cells was counted in each bin from three sections per mouse. At least three mice were examined for each genotype. TUNEL staining was performed following the manufacturer’s protocol (Takara). Cells with a condensed shape and dark black staining were regarded as apoptotic and were easily distinguished from blood cells, which showed no shrinkage and/or were stained brown only in the cytoplasm following the TUNEL protocol.

Cardiac recovery was assessed by measuring the hemodynamic changes using Labchart Pro 8 and Power Lab Data Acquisition System (AD Instruments, AD Instruments Pvt. Ltd, Sydney, Australia). The heart rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and rate pressure product (RPP) were determined. Creatinine kinase myocardial band (CKMB) level was measured using the CKMB kit-14410005 from Agappae (Kochi, India). Triphenyl tetrazolium chloride (TTC) staining and H&E staining were carried out as previously described. Apoptosis was analysed using TUNEL staining (Takara Bio Inc., San Jose, CA, USA) as per the manufacturer’s instructions and triphenyl tetrazolium chloride (TTC) staining was carried out for injury assessment at the structural level. The gene expression of caspases-3,7,9 and PARP were studied using real-time PCR technique as previously mentioned [18 (link)].