Alexa647 conjugated donkey anti rabbit igg antibody

Expanded cells were fixed with 4% paraformaldehyde in 0.1 M PBS for 30 min at 4°C and washed three times. Fixed cells were reacted with blocking solution (PBS with 20% BlockAce [KAC, Kyoto, Japan], 5% BSA [Nacalai Tesque], and 0.3% Triton X-100 [Wako]] for 30 min at room temperature. The following primary antibodies were used: ectodermal marker, neurofilament M (NEFM; 1:200; Millipore, Burlington, MA, USA); endodermal marker, cytokeratin 7 (KRT7; 1:100; Agilent, Santa Clara, CA, USA); and mesodermal cell marker, smooth muscle actin (ACTA2; 1:500: ThermoFisher Scientific). Primary antibodies were diluted in primary antibody dilution buffer (PBS with 5% BlockAce [KAC], 1% BSA [Nacalai Tesque], and 0.3% Triton X-100 [Wako]) and incubated with cells for overnight at 4°C. After three washes, the cells were incubated with Alexa 568-conjugated donkey anti-mouse IgG antibody (1:500: ThermoFisher Scientific), Alexa 647-conjugated donkey anti-rabbit IgG antibody (1:500: Jackson Immuno Research) diluted in secondary antibody dilution buffer (PBS with 0.2% Triton X-100 [Wako]) for 1 h at room temperature. Cells were also stained with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR, USA). Samples were imaged under a fluorescent microscope (BZ-X710; Keyence, Osaka, Japan) at 400x magnification.