Goat anti rabbit igg secondary antibody

Retinal hemispheres containing the injection area were isolated from RCS rats or rd1 mice at PO 2w, 4w, and 8w. Tissue lysis buffer containing 10% PMSF and 90% RIPA was added to extract protein. Proteins (30 μg per well) were separated on a 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% BSA for 1 h at 37 °C, the membranes were incubated with primary antibodies, including rabbit anti-Iba1 (Wako, 016-20001, 1:1000), rabbit anti-GFAP (Abcam, ab48050, 1:2000), mouse anti-GAPDH (Abcam, ab8245, 1:2000), and goat anti-TSPO (Aviva Systems Biology, OALA05219, 1:1000), overnight at 4 °C. The following day, the membranes were washed and incubated with goat anti-rabbit IgG secondary antibody (Invitrogen, #31460, 1:2000) or goat anti-mouse IgG secondary antibody (Invitrogen, 62-6520, 1:2000) or rabbit anti-goat IgG secondary antibody (Invitrogen, 81-1620, 1:2000) for 2 h at room temperature. Finally, the proteins on the membranes were detected using a Pierce™ ECL Western Blotting Substrate (Thermo, 32106) and scanned using a Bio-Rad exposure system (Bio-Rad). Relative protein expression levels were quantified using ImageJ software (NIH) with GAPDH as control. Uncropped images of western blotting and gel are shown in Supplementary Fig. 8.

To analyze the levels of total 4E-BP1 or phosphorylated S6RP and 4E-BP1, unperturbed cells were prepared using same fixation, permeabilization and blocking protocol as for protein synthesis assays. The cells were then incubated o/n in 4°C with 4E-BP1 monoclonal antibody (53H11, Cell Signaling Technology, #34470), p-4E-BP1 monoclonal antibody (Thr37/46, 236B4, conjugated to PE, Cell Signaling Technology, #7547S), p-S6RP monoclonal antibody (S235/236, D57.2.2E, conjugated to Alexa 647, Cell Signaling Technology, #4851S) or isotype specific controls (Rabbit mAb IgG, conjugated to PE or Alexa 647, Cell Signaling Technology, #5742S) in PBS solution containing 5% BSA. All antibodies were used at the concentration recommended by supplier. For analysis of total 4E-BP1 levels, cells were washed and treated with 2 µg/ml secondary antibody (Goat anti-Rabbit IgG secondary antibody conjugated to Alexa Fluor 568, #A-11011, Thermo Fisher Scientific) for 2 hr in RT. Antibodies were washed away using 5% BSA in PBS and the cells were stained for p-Histone H3 (S10) and DNA, as in protein synthesis assays. Finally, the cells were washed three times with PBS, mixed in to PBS supplemented with 1% BSA and put on ice until FACS analysis.