Hrp linked anti rabbit igg antibody

WB analysis was performed as previously described, with some modifications [14 (link),15 (link)]. Cell lysates (protein mass was 10 μg) were loaded onto a 4–15% polyacrylamide gradient gel. To analyze LC3 levels, proteins were incubated on PVDF membranes with rabbit polyclonal anti-LC3 (Cell Signaling Technology, Danvers, MA, USA; #12741, 1:1000) as the primary antibody. The PVDF membranes were incubated overnight at 4°C, washed four times, and incubated with a secondary HRP-linked anti-rabbit IgG antibody (Thermo Fisher Scientific Inc., Waltham, MA, USA; #31458, 1:3000) at room temperature for 1 h. Membranes were washed, immunoreactive proteins were detected, followed by SB analysis. The primary and secondary antibodies were removed from the PVDF membranes using WB stripping solution. Membranes were then washed and blotted with rabbit monoclonal [EPR18351] anti-p62 antibody (Abcam, Cambridge, UK; ab207305, 1:80,000), anti-GA-3 phosphate dehydrogenase (GAPDH) antibody (Abcam; ab8243, 1:10,000), HRP-linked anti-rabbit IgG antibody (1:3000), and HRP-linked anti-mouse IgG antibody (#31432,1:5000). To analyze p62 and GAPDH proteins. PVDF membranes were incubated with anti-p62 and anti-GAPDH antibodies at room temperature for 2 h, followed by HRP-linked anti-rabbit IgG antibody and HRP-linked anti-mouse IgG antibody at room temperature for 1 h.

Mice were anesthetized and transcardially perfused with 0.9% saline. Hippocampal tissues were carefully dissected and then lysed in RIPA buffer (Thermo Fisher) containing 1 mM phosphatase inhibitor, 1 mM protease inhibitor, and 1 mM PMSF (Beyotime Biotechnology) with an ultrasonicator. The proteins were separated on 7.5% SDS–PAGE gels and then transferred onto a polyvinyl difluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% nonfat milk for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C followed by an HRP‐linked anti‐rabbit IgG antibody (31466, 1:3000, Thermo Fisher) for 1 h at room temperature. The primary antibodies used were anti‐β‐actin (AF7018, 1:3000, Affinity) and anti‐NR2B (AF6426, 1:1000, Affinity). The target protein was detected by a ChemiDoc XRS+ system (Bio‐Rad) and then analyzed with ImageJ software (NIH).