Rat ha clone 3f10

Retinas were dissected out from 4% paraformaldehyde (PFA)-fixed eyes and washed extensively in PBS before blocking in staining buffer (10% normal goat serum and 2% Triton X-100 in PBS) for 30 min. Mouse or rabbit antibodies for neuronal class ß-III tubulin (clone Tuj1, 1:500; Covance), rat HA (clone 3F10, 1:200; Roche), phospho-S6-Ser240/244 (1:200, #5364; Cell Signaling), phospho-AKT-Ser473 (1:200, #4058; Cell Signaling), pan AKT (1:200, #2920; Cell Signaling), and phospho-GSK-3β (Ser9) (1:100, #9323; Cell Signaling) were diluted in the same staining buffer. RBPMS guinea pig antibody was made at ProSci, CA, according to publications^{31 (link),32 (link)}. Floating retinas were incubated with primary antibodies overnight at 4 °C and washed three times for 30 min each with PBS. Secondary antibodies (Cy2, Cy3, or Cy5 conjugated) were then applied (1:200; Jackson ImmunoResearch) and incubated for 1 h at room temperature. Retinas were again washed three times for 30 min each with PBS before a cover slip was attached with Fluoromount-G (SouthernBiotech).

Retinas were dissected out from 4% PFA fixed eyes and washed extensively in PBS before blocking in staining buffer (10% normal goat serum and 2% Triton X-100 in PBS) for half an hour. Mouse neuronal class ß-III tubulin (clone Tuj1, 1:500 dilution; Covance) and rat HA (clone 3F10, 1:200 dilution, Roche,) were diluted in the same staining buffer. Floating retinas were incubated with primary antibodies overnight at 4°C and washed three times for 30 min each with PBS. Secondary antibodies (Cy2, Cy3 or Cy5-conjugated) were then applied (1:200–400; Jackson ImmunoResearch) and incubated for 1 h at room temperature. Retinas were again washed three times for 30 min each with PBS before a cover slip was attached with Fluoromount-G (SouthernBiotech). For RGC counting, whole-mount retinas were immunostained with the TUJ1 antibody and 6–9 fields were randomly sampled from peripheral regions per retina to estimate RGC survival. The investigators who counted the cells were blinded to the treatment of the samples. The percentage of RGC survival was calculated as the ratio of surviving RGC numbers in injured eyes compared to contralateral uninjured eyes.