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 euthanized fish examined at 5 wpf was injected with 1 µl EdU solution, while 1.5 µl was used for 6 wpf fish. Fish were fixed 2 hours postinjection 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), fibrillarin (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), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258) were performed at the DF/HCC Research Pathology Core, and FACS at DFCI Flow Cytometry Core according to standard protocols.
Cryo-sectioning and immunofluorescence staining were performed as previously described.44 (link) Antibodies against EGFP (Life Technologies #A6455 and #A11120), fibrillarin (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), HuC/D (Life Technologies #A-21271) and synaptophysin (Millipore #MAB5258) were performed at the DF/HCC Research Pathology Core, and FACS at DFCI Flow Cytometry Core according to standard protocols.
