1832 rbpms

Eyes from mature mice were dissected and fixed for 2 h in 4% paraformaldehyde after puncturing the cornea, washed in PBS, incubated overnight in 30% sucrose at 4 °C, and cryosectioned (14 µm). Right after the eye was dissected-out, a needle was inserted through the cornea on a temporal side of the eye in dorsal to ventral direction, with the needle hub at the dorsal side. After fixation the needle was removed and the eyes were embedded in OCT mold with the eye identity (left or right) and orientation (ventral facing one side and dorsal the opposite side in one direction, and in perpendicular direction temporal and nasal orientation facing opposite sides) marked on the outside of the mold. Slides were marked accordingly, to preserve the information regarding the orientation of cryosections. The cryosections were immunostained for Zic1 (AB134951, Abcam), an RGC marker RBPMS (1832-RBPMS, PhosphoSolutions), and DAPI (Thermo Fisher Scientific). Alexa fluorophore-conjugated secondary antibodies (Thermo Fisher Scientific) were used for fluorescent microscopy (with AxioObserver.Z1, Zeiss). RBPMS+/Zic1+ and RBPMS+/Zic1− RGCs were quantified from nasal, medial, and temporal retinal regions in sections from dorsal and ventral retina, in left and right eyes from four animals each.

All IHC solutions were made up in PBS + 0.3% Triton-X, and all incubation steps were carried out in a humidified chamber. Following a 1 hr protein block in 5% normal donkey serum at room temperature, slides were incubated overnight at 4°C with primary antibodies, washed twice for 5 min each in PBS, incubated for 2 hr at room temperature with secondary antibodies conjugated to various fluorophores (1:1000, Jackson Immunological Research) and Hoechst (1:10,000, Life Technologies), and washed again twice for 5 min each in PBS before coverslipping with Fluoro-Gel (#17985, Electron Microscopy Sciences). Primary antibodies used include guinea pig anti-RBPMS (1:1000, #1832-RBPMS, PhosphoSolutions), rabbit anti-KI67 (1:250, #MA5-14520, Thermo Fisher Scientific), and rat anti-L1CAM (1:10, #130-102-243, Miltenyi Biotec).

RGCs were cultured for 7 days in vitro (DIV) on 12mm coverslips before being fixed in 4% PFA for 15 mins at room temperature (RT) and then permeabilized in 0.1% Triton X-100 for 5 mins. Cells were then blocked for 1 hour at RT with 5% BSA and 5% normal donkey serum. Primary antibodies incubation with guinea pig anti-RBPMS (pan RGC marker) (1:250 dilution, #1832-RBPMS; PhosphoSolutions, Aurora, CO) and mouse anti-TUJ1 (neuron marker) (1:300 dilution, #MMS-435P; San Diego, CA) were performed overnight in a moist chamber at 4 °C. Cells were incubated with Cy3 conjugated secondary antibody (1:1000 dilution, #706-165-148; Jackson ImmunoResearch Laboratories, West Grove, PA) and Alexa Fluor 488 conjugated secondary antibody (1:1000 dilution, #A-21202; Thermo Fisher Scientific, Waltham, MA) for 1 hour at RT. Stained coverslips were sealed on to glass slides with Prolong Diamond antifade with DAPI (#P36971; Thermo Fisher Scientific, Waltham, MA). Six images per coverslips, in a fixed 3x2 grid, were acquired on a Leica DMi8 inverted microscope system (Buffalo Grove, IL). To determine RGC culture purity, RBPMS staining counts were compared to the overlap of co-labeled and only labeled DAPI positive stained cells. Immunocytochemistry of RGC RBPMS staining was performed from three isolations with a total cell count of n = 11,687.

Retinal flat mounts, after fixation and dissection, were rinsed in PBS and blocked-permeabilized for 1 hr with 20% goat serum and 0.4% Triton X-100, in antibody buffer containing 150 mm NaCl, 50 mm Tris base, 1% BSA, 100 mm l-lysine, and 0.04% Na azide, pH 7.4. Retinas were then incubated overnight at 4 °C in antibody buffer containing primary antibodies, washed with PBS 5 times, incubated with secondary antibodies for 1 hr, and with DAPI for 10 min in antibody buffer at room temperature. Samples were then washed again 5 times with PBS before mounting in Prolong Gold (Thermofisher Scientific, P36930). The same procedure was carried out for sectioned retinas and optic nerves, except that prior to permeabilization and staining, samples were embedded in OCT and 15 μm frozen sections were placed on glass slides. Primary antibodies used for immunostaining included anti-MTP18 (1:100; Abcam, ab198217), anti-Brn3a (1:100; Millipore, MAB1585) and anti-RBPMS (1:1000: PhosphoSolutions, 1832-RBPMS). Secondary antibodies were Alexa Fluor 488-, 555-, or 647-conjugated, highly cross-adsorbed antibodies (1:500; Thermofisher Scientific).