Immunolabeling of Neuronal Cell Types

Immunolabeling was performed as previously described [26] (link). Cells were fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature followed by washes with PBS. After incubating in blocking solution containing 10% FBS, 2% goat or donkey serum, 0.1% Triton X-100, 0.02% sodium azide, fixed cells were incubated overnight at 4°C with the following primary antibodies: rabbit anti-GFP (1∶200, Invitrogen, used to detect YFP-expressing cells throughout the study), rabbit anti-Pax6 (1∶200, Millipore), rabbit anti-neurofilament 145 (NF145)(1∶750, Millipore), rabbit anti-Tubb3 (1∶100, Covance), rabbit anti-Otx2 (1∶50, Abcam), goat anti-Sox2 (1∶50, Santa Cruz Biotechnology), mouse anti-Pou4f1 (1∶100, Millipore), mouse anti-Crx (1∶100, Abnova), mouse anti-NF68 (1∶400, Sigma), mouse anti-Islet1 (1∶5, Developmental Study Hybridoma Bank). After washing extensively with 0.1% Tween in PBS, the cells were incubated with secondary antibodies conjugated with Alexa 488 or Alexa 594 (1∶500, Invitrogen). Fluorescent images were captured using a Nikon E800 microscope equipped with a SPOT II camera or an Olympus FluoView 1000 confocal microscope.