Optimal cutting temperature cryomatrix

Tamoxifen (Tx; Sigma) was reconstituted to 40 mg/ml in corn oil and placed in a sonicating water bath for ≥1 hr until dissolved. For conditional gene deletion and lineage tracing, postnatal day 57 (P57) mice received 300 mg/kg Tx by oral gavage, daily for four consecutive days. Mice were terminally anaesthetized with a 30 mg/kg intraperitoneal (i.p.) injection of sodium pentobarbitone (Ilium) and perfusion fixed with 4% paraformaldehyde (PFA) (wt/vol) (Sigma) in phosphate buffered saline (PBS) at a rate of ~9 ml/min. Brains were removed and sliced into 2 mm thick coronal slices using a 1 mm brain matrix (Kent Scientific), before being immersion fixed in 4% PFA/PBS for 90 min at ~21°C. Tissue was cryoprotected by immersion in 20% sucrose (Sigma) (wt/vol) in PBS overnight at 4°C prior to embedding in optimal cutting temperature cryomatrix (Thermo Scientific) and storage at −80°C. Mice that received Tx from P57 and were perfusion fixed 7 days later are referred to as P57 + 7.