Immunostaining of Retinal Neurons in Fixed Tissue

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).