Goat anti mouse igg h l af488

Goat anti-mouse IgG (H+L) AF488 (ThermoFisher, 1:400), goat anti-rabbit IgG (H+L) AF647 (ThermoFisher, 1:400), goat anti-mouse IgM AF568 (ThermoFisher, 1:400) and IRDye 800CW goat anti-rabbit IgG (LI-COR, 1:5,000)

On the 15th and 45th day of neuronal differentiation, cells were washed with PBS, fixed with 4% para-formaldehyde in PBS (pH 6.8) for 20 min at room temperature (RT), and then washed in PBS with 0.1% Tween 20 (Sigma-Aldrich, Saint Louis, MO, USA) three times for 5 min. Nonspecific antibody sorption was blocked by incubation in blocking buffer (PBS with 0.1%, Triton X-100, and 5% fetal bovine serum (HyClone, Waltman, MA, USA)) for 30 min at RT. Primary antibodies Rabbit anti-tyrosine hydroxylase (TH), Mouse anti-βIII Tubulin, and Rabbit anti—Sox1 (all from Abcam, Cambridge, UK) were applied overnight at 4 °C and then washed in PBS with 0.1% Tween 20 three times for 5 min. The secondary antibodies Goat anti-Rabbit IgG (H + L), AF546, Goat anti- Mouse IgG (H + L), AF488 (all from ThermoFisher, Waltham, MA, USA), were applied for 60 min at RT, then washed in PBS with 0.1% Tween 20 three times for 5 min. After that, the cell cultures were incubated with 0.1 μg/mL DAPI (Sigma-Aldrich, Saint Louis, MO, USA) in PBS for 10 min for visualization of the cell nuclei and washed twice with PBS. The cells were investigated using an AxioImager Z1 fluorescence microscope (Carl Zeiss, Oberhohen, Germany), and images were taken with AxioVision 4.8 software (Carl Zeiss, Oberhohen, Germany).