Mouse anti pou4f1

Staged mouse embryos were dissected and immediately fixed in 4% paraformaldehyde (PFA) in PBS at 4°C for 2–3 hours. Samples were embedded and frozen in OCT medium (Tissue-Tek) after dehydration in graded sucrose and sectioned at 14 µm thickness. Before adult retina samples were harvested, vascular perfusion was performed to eliminate the blood remain in the retinal vessels, and then retinas were dissected and fixed in 4%PFA. Retinal flat mount immunostaining was performed as previously described [52] (link). Dilution and sources of antibodies used in this study were: mouse anti-POU4F1 (1∶500, Santa Cruz), goat anti-POU4F2 (1∶500, Santa Cruz), mouse anti-Calbindin (1∶5000, Sigma), rabbit anti-Calretinin (1∶2000, Oncogene), rabbit anti-CHAT (1∶200, Chemicon), sheep anti-CHX10 (1∶200, Exalpha), mouse anti-PAX6 (1∶200, DSHB). Mouse anti-Cyclin D3 (1∶100, Santa Cruz). Alexa-conjugated secondary antibodies (Molecular Probes) were used at a dilution of 1∶1,000. Images were captured with a Zeiss 510 META confocal microscope.

Fish embryos were fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) at 4°C overnight for further analysis. Whole-mount mRNA ISHs were performed as described previously
[54 (link)]. Embryonic mouse brains were dissected in cold PBS and fixed in 4% paraformaldehyde overnight for immunostaining or RNA ISH. Brains were cryoprotected and embedded in optimal cutting temperature (OCT) compound (TissueTek, Torrance, CA, USA) and sectioned in 20 μm thickness. Immunohistochemistry and ISH were performed as described previously
[55 (link)]. Detailed protocols are available on the Li lab website
[56 ]. The following antibodies were used in this study: rabbit anti-GFP (Invitrogen, Carlsbad, CA, USA); mouse anti-neurofilament, anti-Pax3, and anti-Pax6 (Developmental Study of Hybridoma Bank, University of Iowa, Iowa City, IA, USA); rabbit anti-GABA (Sigma); mouse anti-Pou4f1 (Santa Cruz, Biotechnology, Dallas, TX); rabbit anti-Dbx1, a gift from Y. Nakagawa (University of Minnesota, Minneapolis, MN, USA)
[17 (link)]; Alexa secondary antibodies (Invitrogen). To compare the spatial expression domain of molecular markers, ISH and immunohistochemistry were performed on serial sections of the same embryo or carefully matched sections of different embryos.