Peroxidase labeled anti rabbit igg

Extraction of total protein from the cells was processed using the Pierce^® RIPA Buffer (Thermo Scientific, Rockford, IL, USA) in accordance with the protocol provided. All protein concentrations were determined with the Pierce^® BCA Protein Assay Kit. Protein samples were diluted to 10 μg/μl, then 1 μl was slowly spotted onto a 0.2μmImmun‐Blot^® PVDF membrane (BioRad Laboratories, Hercules, CA, USA) and air‐dried. To block nonspecific binding sites, the membranes were incubated using 5% Blotting‐Grade Blocker (BioRad Laboratories) for one hour at room temperature. They were then incubated using Anti‐MT‐3 antibody (product no. ab76618; Abcam, Cambridge, UK), and with Anti‐β‐actin (product no. ab8227; Abcam). The antibody was diluted 1:1000 using 5% Blotting‐Grade Blocker (BioRad Laboratories).
The membranes were then washed four times in PBS (Sigma‐Aldrich, St. Louis, MO, USA) for five minutes each, before being incubated with a secondary antibody (Peroxidase‐Labeled Anti‐Rabbit IgG (product no. PI‐1000; Vector, Burlingame, CA, USA)). This was diluted 1:2000 using 5% Blotting‐Grade Blocker. After washing in PBS, all blots were visualized on photosensitive film. Densitometric analyses of the membranes were performed using GeneSnap (Syngene, Cambridge, UK).

Equal amounts of protein (30 μg) were separated by electrophoresis in precast 4–12% Bis-Tris Gels (Bio-Rad) and transferred to activated/pre-wetted polyvinylidene difluoride membranes. The membranes were hybridized with the following primary antibodies as indicated: AT8 anti-P-tau pSer202/Thr205 (1:500, Thermo Scientific, #MN1020); anti-P-tau pS396 (1:1000, Abcam, #ab109390); anti-total tau T46 (1:1000, Thermo Scientific, #13-6400); anti-GAPDH (1:2000, Santa Cruz, #sc-32233); anti-P-CaMKII (1:1000, Abcam, #ab32678); anti-PSD-95 (1:1000, Millipore, #7E3-1B8), C1q (1:1000, Abcam, #ab182451). Secondary antibodies included: peroxidase-labeled anti-mouse IgG (1:2000, Vector Laboratories); peroxidase-labeled anti-rabbit IgG (1:2000, Vector Laboratories); peroxidase-labeled anti-rat IgG (1:2000, Vector Laboratories). ECL (Pierce®) was used to reveal the immunoreactive proteins, and images were acquired using a Fujifilm ImageReader LAS-4000. Membranes were stripped using a stripping buffer (Thermo Scientific) when required. Luminescent immunoreactive protein bands were quantified using Fiji software (ImageJ).