Perfusions:
Following completion of the experiment, animals were transcranially
perfused. All animals received a lethal dose of sodium phenobarbital
(2ml/kg, Euthatal, Marial Animal Health, UK) administered by
intraperitoneal injection. Once completely unresponsive, the animals
were transcardially perfused with 0.1M phosphate-buffered saline (PBS)
and 4% paraformaldehyde in 0.1M PBS (PFA). The brains were further
post-fixed in PFA for at least 2 hours, and then placed in 25% sucrose
solution for a minimum of 24h. A freezing microtome (8000 Sledge
Microtome, Bright Instruments) was used to cut the brain in 40μm coronal
sections, saved as four simultaneous series. The sections were stored in
cryoprotectant (30% sucrose, 1% polyvinyl pyrrolidone, 30% ethylene
glycol in PBS) in a freezer at -20⁰C until further processing.