Preparation and cryosectioning of fixed mouse brains

Remove fixed brain from 50 mL sample container and discard 4% paraformaldehyde solution into appropriate waste disposal stream.

Place brain back into the 50mL sample container and fill with 30% sucrose in 1x PBS containing 0.01% sodium azide.

Place container on vertical rocker at 4°C for 48h 0m 0s or until brain no longer floats in sucrose solution.

Half fill small metal ramekin (~100mL capacity) with isopentane (2-methyl-butane) and place on dry ice, allowing it to cool to -45°C.

Use plastic tweezers to remove brain from sucrose and immerse in isopentane solution for 0h 0m 15s-0h 0m 20s.

• Monitor solution temperature to ensure it does not drop below -60°C, as temperatures lower than this can cause tissue to crack and fragment.
• Use tweezers lacking teeth, as these will damage/mark the cortex.

Remove frozen mouse brains and either'

• Section immediately, or
• Wrap in foil, then wrap in tissue paper, then place in labelled sample tube -80°C for storage.

If brain tissues are stored at -80°C, move them to -20°C prior to sectioning.

Label 12 well microtiter plates with appropriate identifiers (brain tissue IDs, series #s, etc.) and fill each well ⅔ full with anti-freeze solution.

Cool freezing microtome sample holder with dry ice.

Squeeze OCT onto microtome specimen head until the diameter is approximately the diameter of a 1.5 cm piece.

Allow OCT to freeze (turns opaque from translucent) then shave layers off by cutting with microtome blade until you have created a flat base, the thickness of which should be a little more than 4 mm -5 mm .

Mount caudal end (cerebellum/brainstem) of frozen brain onto flat OCT base using additional OCT compound and non-serrated tweezers.

Crush dry ice and surround the OCT embedded brain, pressing gently to compact the dry ice and leaving only the most rostral aspect exposed to begin cutting (olfactory bulb/pre-frontal cortex).

Section 30 µm tissue sections.

• Ensure microtome blade and clamp are slightly wet, this helps to avoid tissue crumpling or sticking to blade.
• Sections must be transferred quickly into wells containing anti-freeze solution using a paint brush.
• If tissue becomes too soft or loses its visual homogeneity due to thawing (sections will start to shred) pause sectioning, top up crushed dry ice around brain sample holder, wait to re-freeze and continue.
• To collect 12 section series', transfer sections sequentially into 12 labelled vials - section 1 is placed into vial 1, section 2 into vial 2, and so on until the last vial is reached, at which point the process begins again and the next section is placed into vial 1 again.

Once all sections have been cut for a given brain tissue, store sections in anti-freeze solution at -20°C.