A 11120

Eyes were harvested and fixed in 4% paraformaldehyde for 2 h. Following 3 brief washes in 1× PBS, they were embedded in Tissue-Tek OCT compound, frozen and stored until ready for sectioning. Eyes were sectioned at a thickness of 12 μm using a cryostat and mounted on glass slides. Slides were dried for 30 min at 37°C on a slide warmer. Slides were rehydrated in 1× PBS and pre-treated with 0.1% Triton X-100/1× PBS for 10 min. Sections were then incubated in blocking buffer (10% goat serum [Gibco, #16210064, New Zealand origin] in 0.1% Triton X-100/1× PBS) for 2 h at room temperature (RT) in a dark and humidified chamber. Sections were then incubated overnight with primary antibodies reconstituted in blocking buffer (mouse anti-GFP, 1:500, Invitrogen #A-11120; rabbit anti-mCherry, 1:500, Abcam #ab167453). Following three washes in 1× PBS, sections were incubated with secondary antibodies (anti-mouse AlexaFluor488, 1:1000, Invitrogen #A28175; anti-rabbit AlexaFluor594, 1:1000, Invitrogen #A32740) for 2 h at room temperature. Sections were washed in 1× PBS and mounted with Pro-Long Diamond Antifade medium containing DAPI (Life Technologies #P36961). Images were acquired at × 25 magnification using a Leica TCS SP8 confocal microscope (Leica Microsystems).

For EdU pulse labeling, EdU from the Click-iT Alexa Fluor 647 Imaging Kit (Life Technologies #C10340) was diluted to 2.5 mg/ml for retro-orbital injection. Each killed fish examined at 5 wpf was injected with 1 μl EdU solution, whereas 1.5 μl was used for 6 wpf fish. Fish were fixed 2  h post injection for cryo-sectioning; EdU detection was performed according to the manufacturer’s protocol.
Cryo-sectioning and immunofluorescence staining were performed as previously described.^{44 (link)} Antibodies against EGFP (Life Technologies #A6455 and #A11120), Fib (Abcam #ab4566) and activated caspase-3 (BD Biosciences #559565) were used as primary antibodies. Secondary antibodies were conjugated with Alexa 488, 568, 647 (Life Technologies). DAPI (Life Technologies #S36973) was used for nuclear staining. Fluorescent images were taken by a Leica SP5X scanning confocal microscope at the Confocal and Light Microscopy core facility at Dana-Farber Cancer Institute.
Paraffin sectioning, H&E staining and immunohistochemistry with primary antibodies against TH (Pel-Freez #P40101, Rogers, AR, USA), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258, Billerica, MA, USA) were performed at the DF/HCC Research Pathology Core, and fluorescence-activated cell sorting at DFCI Flow Cytometry Core according to standard protocols.