Nah2po4.2h2o

Unless stated, all reagents were sourced from Sigma Aldrich Ltd. (Poole, United Kingdom [UK]), while solvents were purchased from Thermo Fisher Scientific (Loughborough, UK). One-litre of PBS (0.02 M, pH 7.2) consisted 0.876 g NaH₂PO₄.2H₂O (VWR International Ltd., Lutterworth, UK), 2.56 g Na₂HPO₄.2H₂O (VWR International Ltd) and 8.77 g NaCl. Culture media, PBS and water were sterilised by autoclaving at 121 °C for at least 15 min. Antibiotic solutions were sterilised by passing through a sterile polyethersulfone 0.22-μm filter (Millipore, Watford, Herts, UK).

At P67 ± 2, mice underwent terminal anaesthesia via intraperitoneal injection of 100 µl sodium pentobarbitone. This was followed by transcardial perfusion using 0.9 % heparinized saline (until the fluid exiting the cut right atrium was entirely clear), followed by 20–30 ml of 4 % paraformaldehyde dissolved in 0.1 M phosphate-buffered saline (PBS, 0.1 M NaH₂–PO₄·2H₂O, 0.1 M Na₂HPO₄·12H₂O, 0.15 M NaCl, all reagents purchased from VWR International, Poole, UK). Brains were rapidly removed from the skull and post-fixed in 4 % paraformaldehyde (PFA) for ~23 h, then cryoprotected by immersion in 30 % sucrose for ~72 h, before freezing them on dry ice and storing them at −80 °C until sectioning. A 1 in 4 series of coronal sections (20 µm) were cut using a cryostat (−22 °C, Bright Instruments Ltd., Huntingdon, UK) and the free-floating sections stored (−20 °C) in antifreeze solution (0.1 M NaH₂PO₄·H₂O, 0.05 M Na₂HPO₄, 0.15 Mm NaCl, 50 % v/v ethanediol, 1 % w/v polyvinylpyrrolidone, 0.1 % w/v NaN₃, VWR International).