Immunofluorescence Staining of Samd14, HA, and CD117

Cells were cytospun at 2 × 10⁵ cells/ml of 50% FBS/PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Slides were washed with PBS, permeabilized with 0.2% Triton X-100 for 10 min at room temperature, and blocked with BlockAid Blocking solution (Invitrogen) for 1 h at room temperature in a humid chamber. Primary antibodies Samd14 (4 (link)), HA (Cell Signaling Technologies), and CD117/c-Kit (2B8; eBiosciences) were diluted in blocking solution and incubated overnight at 4 °C in a humid chamber. After washing with PBS-T (0.05% Tween 20), the slides were incubated with Alexa Fluor 594 donkey anti-rabbit (Invitrogen) or Alexa Fluor 647 goat anti-rat IgG (Invitrogen) for 1 h at room temperature, washed, and mounted with coverslip using Vectashield mounting media with DAPI (Vector Laboratories). Confocal images were acquired using the LSM800 (Carl Zeiss). All images were analyzed using Zen Software (Carl Zeiss).