Retrieve frozen mouse brain stored at -80°C.

Place brain in cryostat. Set chamber temperature to -16°C to -20°C. Set brain holder temperature to -20°C. Let brain equilibrate to temperature for at least 0h 20m 0s before sectioning.

Insert chuck and blade into cryostat.

Apply a drop of OCT on the chuck, and affix brain vertically (olfactory bulbs up).

Apply another layer of OCT at the base of the brain, turning the brain-holder with the other hand. Cover the cerebellum and the junction between the cerebellum and cortex with OCT. Allow OCT to freeze solid (~0h 5m 0s).

Cut 16 to 30 micron sections. Adjust angle of medio-lateral and ventro-dorsal axis so that sections are free of cutting artifacts, symmetrical, and well-aligned to reference atlas.

Use of anti-roll plate is optional and can help reduce rolling of sections, but must be carefully adjusted to avoid cutting artifacts.

For immunohistochemistry we typically cut 30-micron sections for best results. For RNAscope we typically cut 16 to 20-micron sections.

Collect free-floating sections in 48-well plate.

Label your plate lid and base with brain/experiment details using lab marker and/or lab tape.

Pre-fill wells with 1mL 1X PBS containing 0.01% sodium azide (100mg/L).

Can use syringe with blunt needle to collect sections after sectioning. Dip needle into PBS and collect section by gently touching it on the side of the section.

Collect 5 sections per well. Or for experiments that require finer resolution of coordinate can collect sections serially, adding 1 section per well until the end of the plate and start back at well #A1.

Use parafilm to seal the lid to the plate (air-tight) and refrigerate at 4°Cuntil further processing.