The aortic rings were equilibrated in Krebs buffer and maintained under a 1-g tension for 60 ~ 90 min, with three changes of buffer, before the experimental procedures began.
The peptides were eluted at a flow rate of 1 mL min−1 with a linear gradient of 5%% buffer B (25 mM NaH2PO4 and 1 M KCl in 25 % ACN, pH 2.7) for 7 min, 5 60 % buffer B for 20 min and 60 100 % buffer B for 2 min and maintained in 100%% buffer B for 1 min before equilibrating with buffer B for 10 min prior to the next injection.
The samples were loaded at 8 μL min−1 for 4 min; then eluted at 300 nL min−1 with a linear grandent of 5%% buffer D (95 % ACN, 0.1%% formic acid) for 5 min, 3 35 % buffer D for 35 min, 35 60 % buffer D for 5 min, 60 80 % buffer D for 2 min and maintained in 80 % buffer D for 2 min, finally returned to 5%% buffer D within 1 min and maintained in 5 % buffer D for 10 min.
During this period, the hearts were immersed in buffer and maintained at 39.0°C in a temperature-controlled chamber.
The chamber stage was buffered with 5% CO2/95% air mix and maintained in a humid environment.
MCF-7 cells were cultured in Eagle's Minimum Essential Medium (EMEM), supplemented with 5% foetal calf serum (FCS), 2 mM L-glutamine, and 0.06 % Hepes buffer, and maintained at 37°C in a humid atmosphere of 5% CO2 in air.
Finally, the last supernatant was discarded, and the pellet was re-suspended and maintained in buffer III (sucrose 100 mM, KCl 65 mM, K+-HEPES 10 mM and EGTA 50 μM pH 7.2) to a protein concentration of 0.5 mg/mL for subsequent analyses.
Finally, the last supernatant was discarded, and the pellet was resuspended and maintained in buffer III (sucrose 100 mM, KCl 65 mM, K+-HEPES 10 mM, and EGTA 50 μM pH 7.2) to a protein concentration of 0.5 mg/mL for subsequent analyses.
After they were fixed in 4% PFA, post-fixed in EtOH/acetic acid for 5 min at −20 °C and maintained in equilibration buffer for 1 min, they were incubated with the TdT enzyme in a reaction buffer for 1 h at 37 °C (Apoptosis detection kit, Chemicon, Millipore Ibérica, Madrid, Spain).
Wild-type zebrafish (strain AB) were raised and maintained in E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.00001% Methylene Blue) under standard conditions (28.5°C and 14 hr/10 hr of light/dark cycles) [25].
