Sixteen mice were anesthetized by an intraperitoneal injection of 0.1 mL of 20% w/v sodium pentobarbital (Euthatal, Merial Animal Health Ltd. or Pentoject, Animalcare Ltd.). After complete anesthesia, 10 mL of phosphate buffered saline (PBS; Oxoid) were perfused transcardially, followed by 10 mL of 4% v/v paraformaldehyde (PFA; Alfa Aesar). Whole brains were dissected out and post-fixed for 3–4 h at 4 °C in 4% PFA, and then cryoprotected for 3 days at 4 °C in 30% sucrose solution (w/v in 1 × PBS; VWR Chemicals). Brains were then embedded into optimal cutting temperature (OCT) medium within a cryomould and frozen by placing the mould in isopentane cooled down with liquid nitrogen. Brains were then sectioned, with a thickness of 18 μm, using an NX70 Thermo Fisher cryostat, and cryosections were mounted on Superfrost Plus glass slides (Thermo scientific) and stored at − 80 °C.
Sections were washed for 5 min in PBS, incubated for 15 min in 1 µg/mL DAPI (Sigma), washed and mounted using home-made MOWIOL (Calbiochem) containing 2.5% anti-fading agent DABCO (Sigma-Aldrich), covered with a coverslip (thickness #1.5, VWR international) and imaged the following day.
Sections were washed for 5 min in PBS, incubated for 15 min in 1 µg/mL DAPI (Sigma), washed and mounted using home-made MOWIOL (Calbiochem) containing 2.5% anti-fading agent DABCO (Sigma-Aldrich), covered with a coverslip (thickness #1.5, VWR international) and imaged the following day.