Mingyi Zhou, John Bear, Paul A. Roberts, Filip K. Janiak, Julie Semmelhack, Takeshi Yoshimatsu, Tom Baden

 Method Details

 Tissue preparation, immunolabeling, and imaging

For immunohistochemistry, larvae were euthanized by tricaine overdose (800 mg/l) and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 minutes at room temperature before being washed in calcium-negative PBS. Retinae were then incubated in permeabilization/blocking buffer (PBS with 0.5% Triton X-100 and 5% normal donkey serum) at 4°C for 24 hours, and thereafter transferred to the appropriate labeling solution. For nuclear labeling, tissue was incubated at 4°C in blocking solution with Hoechst 33342 nuclear dye (Invitrogen, H21492, 1:2000) for 24 hours. For membrane staining, tissue was incubated at 4°C in blocking solution with BODIPY membrane dye (Invitrogen, C34556, 1:1000) for 24 hours. For immunostaining, tissue was incubated at 4°C for 72 hours in primary antibody solution (chicken anti-GFP (AbCam, 13970, 1:500), rabbit anti-cox iv (AbCam, 16056, 1:500), diluted in permeabilization/blocking solution). Samples were rinsed three times in PBS with 0.5% Triton X-100, then transferred to secondary antibody solution (donkey anti-chicken IgG CF488A conjugate (Sigma, SAB4600031, 1:500), donkey anti-rabbit IgG CF568 conjugate (Sigma, SAB4600076, 1:500)), diluted in permeabilization/blocking solution and incubated at 4°C for 24 hours. Finally, samples were rinsed three times in PBS with 0.5% Triton X-100 before being mounted in mounting media (VectaShield, Vector, H-1000) for confocal imaging.

GABA immunostaining was performed using rabbit anti-GABA (Sigma, A2052, 1:500) according to the protocol described in [

Ptf1a is expressed transiently in all types of amacrine cells in the embryonic zebrafish retina.