Goat anti pou4f2

Animals were perfused transcardially with 4% (w/v) paraformaldehyde (PFA) in phosphate buffered saline (PBS). Harvested tissues were then fixed in PFA overnight (brain, embryo) or 2 hours (eye). Following fixation of eye tissue, the anterior segment and lens was removed to expose the retina. Retina, brain, and embryo samples were then equilibrated in 30% (w/v) sucrose in PBS and embedded in Tissue Freezing Medium (TFM) compound for sectioning. Retina sections were obtained on slides at 20 μm thickness while brain and embryo sections were obtained as floating sections in cold PBS at 40 μm thickness. For whole mount imaging, embryos were fixed and collected in PBS containing DAPI. The following primary antibodies were used: Guinea Pig Anti-LHX9 (1:20,000; Gift from Dr. Jane Dodd, Columbia University), Chick Anti-GFP (1:1000; Abcam), Rabbit Anti-ER (1:200; Thermo Scientific), Rabbit Anti-RFP (1:1,000; Rockland), Mouse Anti-PAX6 (1:200; DSHB), Mouse Anti-POU4F1 (1:500; SantaCruz), Goat Anti-POU4F2 (1:500; SantaCruz), Mouse Anti-Syntaxin (1:1,000, Sigma Aldrich), Goat Anti-SOX2 (1:500; Chemicon). Alexa-conjugated secondary antibodies (Molecular probes) were used at 1:1,000. Images were obtained on a Zeiss LSM510 confocal microscope or Zeiss Lumar Stereoscope.