Sections at cryostat:

• Thickness 30 µm
• Sections picked up with a wet brush (not too wet) and moved to the TBS-T wells (usually serial sections). Store section in anti-freeze solution in -20°C if not immediately staining.

Transfer the sections into the net-wells.

Washes: 3-step wash, 0h 10m 0s each, on a shaker, in TBS-T. (Between washes, just move

the inserts from one tray to another.)

Blocking step: add blocking solution into a 6-well plate.

Transfer the sections with a clean brush from the net-inserts into the 6-well plate.

Incubate on the shaker for 1h 0m 0s at Room temperature.

Primary antibody: Transfer the sections into a 6-well plate containing the primary antibody solution (1 ml per well).

Incubation: on a shaker in the cold room 1h 0m 0s.

Washes: a 3-step wash, 0h 10m 0seach, in TBS-T.

Secondary antibody: Transfer the sections into a 12-well plate containing the secondary antibody. The secondary antibody will be incubated atRoom temperature.

Incubation: on a shaker, at Room temperature, for 1-2 hours.

Washes: 3-4 times, for 0h 10m 0s, in TBS-T.

DAPI: incubate with DAPI (1:10,000) shaking for0h 10m 0s at Room temperature.

Washes: a 2-step wash, on a shaker, atRoom temperature, 0h 10m 0s each.

Mounting: Mount sections and let them dry for 0h 10m 0s atRoom temperature.

Coverslip: Let the slides dry at least 0h 10m 0sat Room temperature.