Af2948

Coronal slices were dissected out after the indicated reperfusion time and the right hemisphere (the infarct/ischemic side) was selected. For western blotting, proteins were extracted from mouse brains using lysis buffer (50 mM Tris–HCl, pH 7.4, 0.5% Triton X-100, 4 mM EGTA, 10 mM EDTA, 1 mM Na₃VO₄, 40 mM Na₂P₂O₇·10H₂O, 50 mM NaF, 100 nM calyculin A, 50 µg/mL leupeptin, 25 µg/mL pepstatin A, 50 µg/mL trypsin inhibitor, and 1 mM dithiothreitol) as previously described^{72 (link)}. The protein concentrations were quantified using Bradford’s assay and normalized. Equal quantities of protein were loaded and separated by 4–12% SDS–polyacrylamide gels (SDS/PAGE) and transferred to polyvinylidene difluoride (PVDF) or nitrocellulose membranes. Membranes were probed with primary antibodies overnight at 4°C and then with appropriate secondary antibodies. Thereafter, membranes were detected with an enhanced chemiluminescence (ECL) immunoblotting detection system (Amersham Biosciences, NJ, USA) using Luminescent Image Analyzer (LAS-4000 mini, Fuji Film, Tokyo, Japan). The densities of the bands were analyzed with ImageJ software (NIH, Bethesda, MA, USA). Primary antibodies were: SAA (R&D systems, AF2948); transferrin (Abcam, ab84036); NLRP3 (Abcam, ab214185); actin (Abcam, ab179467). Secondary antibodies were: Goat anti-rabbit (Sigma, A6154).