Brn3b

Cells were immunolabeled as described previously [28 (link)]. Briefly, EBs and cell samples were fixed in 4% paraformaldehyde for 10–15 min, permeabilized with 0.1% Triton X-100/PBS (1X; 140 mM NaCl, 10 mM KCl, 8 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4; Thermo Scientific, Rockford, IL) for 10 min, blocked in 4% bovine serum albumin (BSA) for 30 min, and incubated with primary antibodies overnight at 4 °C. The next day, the samples were washed three times with PBS and subsequently incubated with Alexa Fluor 488 or 555 labeled secondary antibody (1:300, Invitrogen) for 30 min at room temperature in the dark. After washing three times with PBS, the samples were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, 1 μg/ml; Molecular Probes, Carlsbad, CA). Primary antibodies Nanog (Cell Signaling, Danvers, MA), Oct3/4, Pax6, Nestin, Tuj, Chx10 (all from Millipore, Temecula, CA), Sox2, ZO1, Rx, calretinin, iSlet1, synaptophysin (all from Abcam, Burlingame, CA), Otx2 (Invitrogen), Chx10 (Santa Cruz, Dallas, TX), and Rx (Santa Cruz) were used at 1:200 dilution. Pax6 (DSHB, Iowa City, IA) was used at 1:50 dilution. K_i-67 (Abcam) and Brn3b (Abcam) were used at 1:1,000 dilution. Fluorescent confocal images were acquired using a laser scanning microscope (LSM 510; Carl Zeiss, Thornwood, NY).

Cells were trypsinized into single cell suspensions and then fixed in Fixation Medium Reagent A (Invitrogen; 1×10⁶ cells) for 15 min, permeabilized with Permeabilization Medium Reagent B (Invitrogen), and stained with primary antibodies Chx10 (Abcam, 1:500 dilution) or Brn3b (Abcam, 1:1,000 dilution) for 20 min at room temperature, and washed once in PBS + 0.1% NaN₃ + 5% fetal bovine serum (FBS). Then the cells were incubated with fluorescein isothiocyanate (FITC)–conjugated secondary antibodies (anti-rabbit FITC, Invitrogen, 1:300) for another 20 min in the dark, washed and resuspended in PBS buffer, and analyzed with flow cytometry (FACS Aria; BD Biosciences, Franklin Lakes, NJ).