Phalloidin and nucleus staining of chicken embryonic skin

Embryos were fixed in 4% (w/v) paraformaldehyde (PFA) at 4 °C for 12 hours, and a piece of the skin was peeled from the flank region of embryos. Specimens were treated with PBST (phosphate-buffered saline with 0.1% (v/v) Tween 20) at 4 °C. To detect filamentous actin and nucleus, specimens were subjected to a reaction with Alexa Flour 568 phalloidin (Thermo Fisher), and 4′, 6-Diamidino-2-phenylindole (DAPI) (DAPI solution 1 mg/ml, Dojindo) (phalloidin 1/300 and DAPI 1/1000 dilution in PBST) for 14 hours at room temperature. After washing in PBST at room temperature, specimens were sealed by Fluor Save reagent. Microscopic images were obtained using the confocal laser-scanning microscopy Nikon A1R.