Mouse neuronal class β 3 tubulin clone tuj1

Retinas were dissected out from 4% PFA fixed eyeballs and washed extensively in PBS before blocking in staining buffer (10% normal goat serum (NGS) and 2% Triton X-100 in PBS) for half an hour. All antibodies were diluted in the same staining buffer. Antibodies used were: mouse neuronal class β-III tubulin (clone Tuj1, 1:500 dilution; Covance), rat HA (clone 3F10, 1:100 dilution, Roche), pS6 rabbit antibody (1:400, Cell Signaling). Floating retinas were incubated with primary antibodies overnight at 4°C and washed 3 times for 30 minutes each with PBS. Secondary antibodies (Cy2, Cy3 or Cy5-conjugated) were then applied (1:200–400; Jackson ImmunoResearch) and incubated for 1 hour at room temperature. Retinas were again washed 3 times for 30 minutes each with PBS before a cover slip was attached with Fluoromount-G (Southernbiotech).

4% PFA fixed eyes were dehydrated with increasing concentrations of sucrose solution (15%–30%) overnight and embedded in OCT on dry ice. Serial cross sections (12 μm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The retina sections were blocked in the staining buffer (10% normal goat serum (NGS) and 0.1% Triton X-100 in PBS) for 30 minutes followed by primary antibody staining. The following antibodies were used: mouse neuronal class β-III tubulin (clone Tuj1, 1:500 dilution; Covance), phospho-AKT-Ser473 (193H12, 1:200 dilution; Cell Signaling). The sections were incubated with primary antibodies overnight at 4°C and washed three times for 15 minutes each with PBS. Secondary antibodies (Cy2, Cy3 conjugated) were then applied (1:200–400; Jackson ImmunoResearch) and incubated for 1 hour at room temperature. Sections were again washed 3 times for 15 minutes each with PBS before a cover slip was attached with Fluoromount-G (Southernbiotech).