Pregnant females were cervically dislocated and the embryos were removed from the mother, and placed into ice-cold 0.01M phosphate buffered saline (PBS). E12-14 embryos were decapitated, and the brain was dissected and placed into 4% paraformaldehyde (PFA) for overnight fixation. Considering the larger size of the E15 embryos and to ensure adequate fixation we performed cardiac perfusion on E15.5 embryos. The arm of the embryo was removed to reveal the heart and the animal was perfused with 1 ml 0.01M PBS followed by 3 ml 4% PFA. The brain was then removed and fixed overnight in 4% PFA. Brains were then washed in 0.01M PBS for 5 h to remove the fixative. Embryonic tissue was embedded in 4% Bacto-Agar in 0.01M PBS heated to 37°C, allowed to cool, and then sectioned at 60 μm using a Leica 1000S vibratome and kept in a cryoprotective ethylene glycol solution at -20°C until further processing for immunohistochemistry.
Postnatal mice (P0-P30) were deeply anaesthetised on ice (P0-P4) or with 4–4.5% isoflurane (P6-P30) and perfused transcardially with 0.01M PBS followed by 4% PFA. Brains were post-fixed for 2–5 h in 4% PFA. The fixative was then washed from tissues 3 times for 30 min each with 0.01M PBS. Tissue was sectioned at 60 μm using a Leica 1000S vibratome and kept in a cryoprotective ethylene glycol solution at -20°C until further processing for immunohistochemistry.
Postnatal mice (P0-P30) were deeply anaesthetised on ice (P0-P4) or with 4–4.5% isoflurane (P6-P30) and perfused transcardially with 0.01M PBS followed by 4% PFA. Brains were post-fixed for 2–5 h in 4% PFA. The fixative was then washed from tissues 3 times for 30 min each with 0.01M PBS. Tissue was sectioned at 60 μm using a Leica 1000S vibratome and kept in a cryoprotective ethylene glycol solution at -20°C until further processing for immunohistochemistry.