TITLE: Histology Protocol
AUTHORS: Sasha Burwell

[DESCRIPTION]
This protocol details the histological processing of a mouse brain.

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

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2. Deeply anesthetize mouse with isofluorane until completely under (no toe pinch response, low to no breathing).
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3. 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 1 s with a wire attached to the positive port of the battery. This will create a small lesion for histological viewing of electrode placement.

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4. Fix with a transcardial perfusion of 15 mL PBS followed by 50 mL of 4% PFA.
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5. Carefully remove the brain from the skull and post-fix in 50 mL of 4% PFA at 4 °C  .
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6. 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.
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7. The next day, wash with PBS, with a 15 min agitation in a cold room for the first wash (1/3).

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

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9. The next day, wash with PBS, 15 min or longer agitation in a cold room for the remaining washes (3/3).
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10. Embed the brains in 4% agarose.
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11. Using a Leica VT1200S vibratome, slice the brain along the coronal axis at 50 1534 thick slices.

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12. If performing tyrosine hydroxylase immunostaining:
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13. Mount the sections onto SuperFrost glass slides. Let dry completely, then refresh with PBS for 20 min.

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12.1. Place sections in a 24 well plate, 2-3 sections per well.
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12.2. Wash 1x in PBS.
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12.3. Incubate for 120 min in a blocking solution consisting of 5% goat serum, 3% bovine serum albumin, and 0.3% trition-x.
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12.4. Then transfer sections to a half-block solution containing 1:1000 rabbit α-TH, and leave 120 min at 4 °C on a shaker.

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

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12.6. Incubate for 240 minin a half-block solution containing 1:1000 goat α-rabbit 488 .

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12.7. Wash in 0.1 Molarity (M) PBS with 0.1% tween.
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12.8. Wash in PBS.
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14. Rinse slides with MilliQ, then coverslip with 155 µL of Vectashield Vibrance + DAPI mounting medium.

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15. Let set at Room temperaturefor at least 2 hours, and up to 120 min.

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16. When set, image slides on a VS200 slide scanner using the following protocol:
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16.1. Overview in brightfield.
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16.2. Focus in 10% Tritc (make sure focus points are not over the VTA).
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16.3. Image at:

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