Alexa flour 680 donkey anti rabbit

The antibodies used were: mouse anti- CD-9 (C-4, #sc-13118), mouse anti-Alix (1A12, #sc-53540) and goat anti-EGFR (N-20, #sc-31155) from Santa Cruz Biotechnology Inc, (Santa Cruz, CA); rabbit anti-LAMP2 (#ab37024) from Abcam (Toronto, ON) and rabbit anti-GRP94 (#2104) from Cell Signaling Technology (Whitby, ON). All primary antibodies were used at a concentration of 1:1000 for western blot analyses. The secondary antibodies used for detection were Alexa Flour 680 donkey anti-rabbit (# A10043, 1:10000), donkey anti-mouse (#A10038, 1:10000) and donkey anti-goat (#A21084, 1:10000) from Life Technologies (Invitrogen), Burlington ON.

Cardiomyocytes were plated on glass coverslips, fixed with 4% paraformaldehyde for 10 min and blocked with 5% bovine serum albumin in PBS for 1 h at room temperature. Then, the cells were incubated with specific primary antibodies at 4°C overnight and were subsequently incubated with the corresponding secondary antibodies for 1 h at 37°C. The nuclei were stained for 5 min with 4′,6-diamidino-2-phenylindole (DAPI). Cells were imaged using a confocal microscope. The following primary antibodies were used in this experiment: mouse monoclonal anti-P-MLKL (AF7420, Affinity Biosciences), anti-RIPK1 (17519-1-AP, Proteintech Group), anti-RIPK1 (ab72139, Abcam), anti-RIPK3 (374639, Santa Cruz Biotechnology), anti-RIPK3 (ab62344, Abcam) and anti-LC3B (L7543, Sigma-Aldrich). The following secondary antibodies were purchased from Invitrogen: Alexa Flour 488 donkey anti-rabbit (A21206), Alexa Flour 568 donkey anti-mouse (A10037), and Alexa Flour 680 donkey anti-rabbit (A32802).