Alexafluor antibody

Cells were cultured in chamber slides (SPL Life Sciences, Gyeonggi‐do, Korea), fixed with 4% PFA, and permeabilized with 0.25% Triton X100. After blocking in PBS containing 2% bovine serum albumin (Bio‐Rad), cells were incubated with a primary antibody against AXL (Cell Signaling Technology). An isotype‐matched antibody was used as a control. Alexa Fluor antibody (Thermo Fisher Scientific) was used as the secondary antibody. Cell nuclei were visualized with DAPI (Santa Cruz Biotechnology). Images were captured using a confocal microscope (Carl Zeiss LSM880 with Airyscan, Jena, Germany).

Pregnant female mice were injected intraperitoneally with BrdU (100 mg/kg, Sigma-Aldrich, B5002) at the specified embryonic age between ZT9 and ZT10. Embryos and pups were fixed and sectioned as described above. Tissues were treated with 2N HCl for 25 min at 55 °C, washed 3 times in PBS at room temperature, and incubated in 10% heat inactivated horse serum (Wisent Inc.) in PBST for 1 h. The slides were incubated overnight at 4 °C in fresh blocking solution containing rat anti-BrdU antibody (1:1000, Bio-Rad Laboratories, Hercules, CA, USA, OBT0030G) and rabbit anti-MeCP2 antibody (1:200, EMD Millipore, Burlington, MA, USA, 07-013). The next day, tissues were washed 5 × 5 min with PBST and incubated for 2 h at RT in the dark with the appropriate secondary AlexaFluor antibody (1:1000, ThermoFisher Scientific) diluted in blocking solution. Sections were washed 5 × 5 min with PBST, followed by one PBS rinse. Free-floating sections from P0 and P7 animals were mounted onto glass microscope slides. Slides were coverslipped with Vectashield Mounting Medium (Vector Biolabs) and sealed with nail polish.

Tissues were washed 5 × 5 min in PBS (pH 7.4) with 0.1% Triton X-100 (PBST), incubated for 1 h at room temperature (RT) in blocking solution (10% heat inactivated horse serum (Wisent Inc., Saint-Jean-Baptiste, QC, Canada) in PBST), and incubated overnight at 4 °C in fresh blocking solution containing the primary antibody (Goat anti-GFP, 1:1000, Eusera, Edmonton, AB, Canada, EU3; Rabbit anti-SOX2, 1:300, Abcam, Cambridge, UK, ab97959). The next day, tissues were washed 5 × 5 min with PBST and incubated for 2 h at RT, protected from light, with the appropriate secondary AlexaFluor antibody (1:1000, ThermoFisher Scientific, Waltham, MA, USA) diluted in blocking solution. Sections were washed 5 × 5 min with PBST, incubated for 10 min in DAPI (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS, washed twice in PBS, and mounted onto glass microscope slides. Slides were coverslipped with Vectashield Mounting Medium (Vector Biolabs, Malvern, PA, USA) and sealed with nail polish.