Multispectral imaging system

IHC was performed on retina and ON cryo-sections and SC paraffin sections to validate protein expression of Rbpms (RGC survival, n=3–7) and NRN1 and GFP (AAV2-induced overexpression, n=4). Whole eyes with ONs were harvested and fixed in 4% PFA for 2 h at room temperature. After fixation, the tissue was placed in 20% sucrose overnight at 4 °C and embedded in optimum cutting temperature the next day. Sections (10 μm) were cut using a cryostat (Leica Biosystems, Richmond, IL, USA). Cross-sections of retina were transferred to Superfrost glass slides (Fisher Scientific). Slides were incubated in PBS for 10 min and blocked with SuperBlock Blocking Buffer at room temperature for 1 h. Primary antibodies (Supplementary Table S1) were diluted in SuperBlock. Each slide was incubated with the respective primary antibody and incubated overnight at 4 °C. Sections were then washed and incubated with AlexaFluor secondary antibody (Supplementary Table S1) for 1 h at room temperature. After rinsing, slides were mounted with ProLong Gold anti-fade reagent with DAPI. Sections were observed and captured using a Nikon Eclipse Ti-U Microscope containing the Nuance Multispectral imaging system and analyzed using Adobe Photoshop CS5 software.

RNA in situ hybridization was performed to verify effective transduction of AAV2–GFP and AAV2–hNRN1 (n=4). Cryo-fixed retina cross-sections were subjected to protein digestion using proteinase K stock (Panomics, Santa Clara, CA, USA) diluted 1 : 100 for 20 min at room temperature. In situ hybridization was performed using type 1 probes for hNRN1 (VAI-15422) designed by Panomics following manufacturer's protocols. Briefly, probes were diluted in 1 : 50 in hybridization buffer and sections incubated at 40 °C for 3 h and then overnight at room temperature. The sections were then washed and hybridized in succession after each of the following treatment: PreAmp1 QF (1 : 100) at 40 °C (25 min), Amp1 QF (1 : 100) at 40 °C (15 min), Label Probe AP (1 : 1000) at 40 °C (15 min). Sections were finally incubated with AP Enhancer solution (Affymetrix, Inc., Santa Clara, CA, USA) for 5 min at room temperature and Fast Red substrate (chromogenic dye) at 40 °C (30 min). Slides were washed and mounted with ProLong Gold anti-fade reagent with DAPI. Sections (three sections/mouse, n=4) were observed and captured at × 400 magnification using a Nikon Eclipse Ti-U Microscope containing the Nuance Multispectral imaging system and analyzed using Adobe Photoshop CS5 software.

Gap43 and Nefm labeling of RGC axons within the GCL was performed with a modified flat-mounting protocol.^{73 (link), 80 (link)} Two weeks after IVT injection of either AAV2–GFP or AVV2–hNRN1, ONC was performed. At 28 dpc, mice (n=5 per group, AAV2–GFP and AAV2–hNRN1) were killed, eyes enucleated and fixed in 4% PFA (Electron Microscopy Sciences, Hatfield, PA, USA) for 2 h. Post fixation, retinas were carefully dissected and washed several times with PBS. Pre-treatment with 0.3% Triton X-100 was done for an hour at room temperature. Retinas were then soaked in blocking buffer (10% goat serum, 0.3% Triton X-100, 60 min, room temperature) and then incubated with primary antibodies (Supplementary Table S1) diluted in 10% goat serum+0.3% Triton X-100 using a modified protocol with incubation overnight at room temperature. The next day, retinas were left at 50 °C for 4 h and washed multiple times with PBS and incubated with AlexaFluor secondary antibody (Supplementary Table S1) diluted in 0.1% Triton X-100 with gentle agitation overnight at 4 °C. After rinsing, retinas were flat-mounted RGC side up with ProLong Gold anti-fade reagent with DAPI. Images at x400 magnification were observed and captured using a Nikon Eclipse Ti-U Microscope containing the Nuance Multispectral imaging system and analyzed using Adobe Photoshop CS5 software.