Bax inhibiting peptide v5

BAX Inhibiting Peptide V5 (Catalogue Number. B1436) and Pifitrin-α (Catalogue Number. 506132) were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Spinal-cord cultures were exposed to the H₂S donor, Na₂S (SIGMA-Aldrich, Milan, Italy), following the same protocol described in Davoli et al., [1 (link)]. All experiments were performed between 10 and 12 days in vitro (DIV). For imaging experiments, cells were plated on 13-mm cover glasses. At 10 DIV, the cells were pre-exposed to the BAX Inhibiting Peptide V5 (SIGMA-Aldrich, Milan, Italy; 50 µM) or necrostatin-1 (Merck S.p.a., Roma, Italy; 5 and 15 µM) for 1 h, and then co-exposed to 100 µM and 200 µM Na₂S for 18 h. After 18 h, the cells were fixed in paraformaldehyde (4%). To visualize motor neurons, cultures were stained with SMI-32 (Covance, Princeton, NJ, USA; 1:1000), and nuclei were stained with Hoechst 33342 (SIGMA-Aldrich, Milan, Italy). SMI-32-positive cells were quantified by direct counting, and their number was normalized to the untreated spinal-cord preparation. For toxicity experiments, we performed a Student’s t-test, setting the significance to p < 0.05.