Fluor s max imager

Western blot (WB) analysis was performed on protein extracted from post-ONC mouse retinas, primary microglia, and RGCs as previously described [28 (link)]. Washed and blocked polyvinylidene fluoride membranes from WBs were incubated with primary antibodies. Primary antibodies included anti-Sema3A (1:1000, rabbit monoclonal, Abcam, ab23393), anti-CD16/32 (1:1000, goat polyclonal, R&D, AF1460), anti-CD206 (1:500, rabbit monoclonal, Abcam, ab125028), anti-Iba1 (1:1000, rabbit monoclonal, Abcam, ab178846), anti-SMI32(1:1000, rabbit monoclonal, Abcam, ab207176), and anti-Map2 (1:1000, rabbit monoclonal, CST, #4542). Membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, United States) in TBS-T for 1.5 h at room temperature. ECL-Plus reagent (Bioground) was applied to the membranes and chemiluminescence visualized by a Fluor-S-max imager (Bio-Rad).

Activation of the PI3K and MAPK pathways following different treatments was assessed via western blot on the whole retina tissue. Animals were killed, their eyes enucleated, and the retinas immediately extracted in ice-cold PBS. Retinas were individually sonicated in 400-μl solutions of ice-cold SDS lysis buffer (2% SDS, 0.3% DTT, and 10% glycerol in 40-mm Tris-Cl, pH 6.8) prior to denaturing (heated to 90 °C for 8 min) and removal of cellular debris by centrifugation (12,000 rpm, 10 min, 4 °C).
Proteins were separated by SDS-PAGE on 10% acrylamide Bio-Rad TGX gels and then transferred via semidry blotting to a 0.2 -μm pore nitrocellulose membrane. Membranes were blocked with 5% milk in TBS-T for 1 h at room temperature. Primary antibody incubation occurred in 1% milk in TBS-T overnight at 4 °C on a platform rocker. The secondary antibody was HRP-conjugated, and incubation was performed for 1 h at room temperature in 5% milk in TBS-T, on a platform rocker.
Blots were imaged using a Bio-Rad Fluor-S Max imager (Bio-Rad, Mississauga, ON). Loading was verified by re-probing the blots with antisera directed against GAPDH (1:1000; rabbit polyclonal; Cell Signaling Technology). Normalized densitometry values (relative to GAPDH) for each experimental group were averaged ±SEM, and statistical analysis was performed by ANOVA followed by post hoc analysis using Tukey’s post hoc comparisons.