Intracardiac perfusion and rat brain fixation for immunohistochemistry

Before starting, fill the tube of a peristaltic pump with ice-cold 1X PB solution

Deeply anesthetize the animal. Check for no reflexes and slower breathing

Cut the animal's thorax and perfuse the animal through the left ventricle with ice-cold 1X PB solution, after a small cut of the right atrium to allow fluid flux

Remove brains from the skull and divide the two hemispheres into two halves along the medial sagittal line (one hemisphere is freshly dissected and processed for high dimensional flow cytometry, and the other one is processed for histological analysis as follow)

For histological analyses, postfix one brain half in 40 ml formaldehyde solution 4% in PB buffered for 72h, at 4°C (replace every 24 hours with fresh fixative solution)

Rinse three times in PB and immerse in 30% sucrose + 10% glycerol solution at 4 °C until sinking for cryoprotection

Include brains with OCT

Freeze in 2-methylbutane, cooled into liquid nitrogen, and store at -80 °C

Perform sectioning with a cryostat at 30-μm-thickness

Store sections at 4°C in 0.1 M PB containing 0.02% sodium azide before being further processed for immunohistochemical staining