Histology Protocol

Prepare 4% paraformaldehyde in 0.1Molarity (M) PB, and 7.4. Let chill to 4°C.

Deeply anesthetize mouse with isofluorane until completely under (no toe pinch response, low to no breathing).

If the mouse has an electrode bundle implanted, quickly attach their tongue with an alligator clip to the negative port of a 9V battery, and touch each electrode connection for 0h 0m 1s with a wire attached to the positive port of the battery. This will create a small lesion for histological viewing of electrode placement.

Fix with a transcardial perfusion of 15mL PBS followed by 50mL of 4% PFA.

Carefully remove the brain from the skull and post-fix in 50mL of 4% PFA at 4°C .

The next day, wash 3x with PBS, with a 15 min agitation in a cold room for the first wash, and 15 mins or longer agitation in a cold room for the remaining washes.

The next day, wash with PBS, with a 0h 15m 0s agitation in a cold room for the first wash (1/3).

The next day, wash with PBS, 0h 15m 0s or longer agitation in a cold room for the remaining washes (2/3).

The next day, wash with PBS, 0h 15m 0s or longer agitation in a cold room for the remaining washes (3/3).

Embed the brains in 4% agarose.

Using a Leica VT1200S vibratome, slice the brain along the coronal axis at 50 thick slices.

If performing tyrosine hydroxylase immunostaining:

Place sections in a 24 well plate, 2-3 sections per well.

Wash 1x in PBS.

Incubate for 2h 0m 0s in a blocking solution consisting of 5% goat serum, 3% bovine serum albumin, and 0.3% trition-x.

Then transfer sections to a half-block solution containing 1:1000 rabbit α-TH, and leave 2h 0m 0s at 4°C on a shaker.

Wash in 0.1Molarity (M) PBS with 0.1% tween.

Incubate for 4h 0m 0sin a half-block solution containing 1:1000 goat α-rabbit 488 .

Wash in 0.1Molarity (M) PBS with 0.1% tween.

Wash in PBS.

Mount the sections onto SuperFrost glass slides. Let dry completely, then refresh with PBS for 0h 20m 0s.

Rinse slides with MilliQ, then coverslip with 155µL of Vectashield Vibrance + DAPI mounting medium.

Let set at Room temperaturefor at least 2 hours, and up to 2h 0m 0s.

When set, image slides on a VS200 slide scanner using the following protocol:

Overview in brightfield.

Focus in 10% Tritc (make sure focus points are not over the VTA).

Image at:

1. 300ms 50% Cy5
2. 193ms 10% Tritc
3. 100ms 10% Fitc (only if GCaMP or TH present; otherwise, turn off this channel)
4. 20ms 10% DAPI