Cultures were fixed in 4% (w/v) paraformaldehyde (Electron Microscopy Sciences) in 1× PBS for 15 min at room temperature (RT), and washed twice with PBS. Fixed cells were incubated for 1 hr at RT in blocking solution (PBS with 2% (v/v) goat serum (Vector Laboratories), 0.1% (v/v) Triton X-100 (Sigma Aldrich)). Immunostaining was performed overnight at 4°C with primary antibodies diluted in PBS and 0.1% (v/v) Triton X-100. Cells were rinsed three times with PBS and incubated for 1 hr at RT with secondary antibodies, protected from light. Fixed cultures were rinsed once with DAPI in PBS and coverslipped with Fluormount-G (SouthernBiotech). Primary antibodies used: FOXG1 (Abcam, ab18259), TH (Millipore, AB5986); CALB2 (Millipore, MAB1568), CORT (LifeSpan BioSciences, LS-C143717), GAD67 (Millipore, MAB5406), GFP (Nacali Tesque, 04404-84, or Millipore, 06-896), MKI67 (BD Pharmingen, 550609), NR2F2 (R&D Systems, PP-H7147-00 or Abcam, ab64849); SOX2 (Millipore, AB5603); SST (Millipore, MAB354); NKX2-1 (Progen, 16108). Secondary antibodies used were species-specific Alexa Fluor dyes (ThermoFisher Scientific).
