2,3,5-triphenyltetrazolium chloride monohydrate staining

Animals were euthanized at 72 h post-HI and infarct area was assessed using 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining. Briefly, brains were sectioned into a total of five slices/brain with each slice being 2 mm thick. The slices were then immersed in 2% TTC solution until the color turned red (red is indicative of healthy, living tissue and white shows the infarcted tissue), as previously described (Zhou et al., 2009; Li et al., 2012). Slices were washed from TTC solution using PBS and stored in 10% formaldehyde solution overnight. To calculate the percentage infarcted area of total brain area, ImageJ software was used. For statistical analysis, data was presented as mean±s.d. using one-way ANOVA followed by Tukey multiple-comparison post hoc analysis test, showing significance at *P<0.05 versus sham; ^#P<0.05 versus vehicle; ^@P<0.05 versus Ad-TMBIM6; ^$P<0.05 versus BI-1 siRNA; ⁺P<0.05 versus Ad-TMBIM6+scramble siRNA; ^&P<0.05 versus IRE1α CRISPR AP; ^%P<0.05 versus control CRISPR. n=6/group.