Prs2283

Immunostainings for RIP3 and pMLKL were performed using the spinal cord sections obtained 3 days, and those for NeuN and RT97 were performed using sections obtained 42 days after SCI. The sections were washed in PBS for 15 min, after which they were washed with PBS containing 0.3% Tween for 10 min and blocked with 3% milk and 5% FBS in 0.01 M PBS for 2 h. The sections were incubated with rabbit anti-RIPK3 antibody (1:100; PRS2283, Sigma-Aldrich), rabbit anti-phospho-MLKL antibody (1:150; ab196436, Abcam, Cambridge, UK), mouse anti-NeuN antibody (1:100; MAB377, Millipore, Burlington, MA, USA) or mouse anti-RT97 antibody (1:30; AB528399, DSHB, Iowa City, IA, USA) diluted in PBS overnight at 4 °C. After rinsing with PBS, the sections were incubated with goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (1:500; A11034, Invitrogen, Carlsbad, CA, USA), donkey anti-rabbit IgG Alexa Fluor 594 secondary antibody (1:500; A21207, Invitrogen), goat anti-mouse IgG Alexa Fluor 488 secondary antibody (1:500; A11001, Invitrogen), or donkey anti-mouse IgG Alexa Fluor 594 secondary antibody (1:500; A21203, Invitrogen) for 1 h at room temperature. The sections were mounted with Vectashield containing DAPI to label the nuclei (Vector Laboratories). The sections were stained at the same time in each experiment.

According to previous studies, total protein was extracted from rat myocardial tissue and cultured cardiac cells with RIPA lysis buffer supplemented by a protease inhibitor cocktail (Complete, Roche). After quantification using the BCA kit (Beyotime Institute of Biotechnology, China), the equal amount of protein samples were separated by SDS-PAGE and then transferred to the PVDF membrane. The membranes were blocked with nonfat milk in 1X TBST for 1 hour at room temperature and then incubated with the primary antibodies at 4°C overnight. The next day, the membranes were washed using 1 X TBST for 3 × 5 min, and then, the membranes were incubated with secondary antibodies (at an optimized concentration) for 1-2 hours at room temperature. Finally, an image analyzer (Bio-Rad) was used to detect and evaluate the band density on the film. The primary antibodies include against RIP3 (PRS2283, Sigma), β-MHC (MA1-83347, ThermoFisher Scientific), ANP (PA5-29559, ThermoFisher Scientific), MLKL, ZO-1 (40-2300, ThermoFisher Scientific), GAPDH (PA1-987, ThermoFisher Scientific), and ACTIN (ab6276, Abcam). The secondary antibody used including goat anti-rabbit IgG and goat anti-mouse IgG was purchased from Abcam (ab6789, ab205718, Abcam).