Rat brain processing for histological analyses (update)

Deeply anesthetize animals with sodium pentobarbital (50 mg/kg, i.p.)

Perfuse through the left ventricle with saline [0.9% (wt/vol)] at room temperature (RT)

Perfuse again with ice-cold formaldehyde solution 4% in PBS buffered for histology

Remove brains and post-fix them for 24 h in the same fixative

Wash twice with 0.1 M PBS and process for paraffin embedding following standard procedures (performed by an external facility)

Alternatively, brains could be stored in 0.1 M PBS at 4 °C (not for over a month without changing the PBS) prior paraffin processing

Cryoprotect for 24-48 h (until they sink) in 30% sucrose at 4 °C

Exchange sucrose for 0.1 M PBS

Immerse brains in cold (-30ºC) 2-methylbutane for 30 s and store at -80 °C

Include in OCT

Perform sectioning with a sliding microtome at 5-μm-thickness for paraffin samples or

in a cryostat at 20- or 30-μm-thickness for frozen samples.