TITLE: Immunohistochemistry Protocol
AUTHORS: Livia Hecke Morais, Jenny Ji

[DESCRIPTION]
Protocol has been approved by the California Institute of Technology’s Institutional Animal Care
and Use Committee (IACUC).

[STEPS]
SECTION: Brain Extraction
1. Mice were anesthetized with 150 uL pentobarbital (Euthasol), and their hearts were punctured.

SECTION: Brain Extraction
2. Mice were perfused with ice-cold phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA).
SECTION: Brain Extraction
3. Brains were extracted and preserved in 4% PFA for 48 hours while shaking at 4°C. 
SECTION: Brain Extraction
4. Brains were then transferred to PBS + 0.05% sodium azide for storage.
SECTION: Preparation for Brain Slicing
5. To prepare PBS with 2% agarose and 0.02% sodium azide:
SECTION: Preparation for Brain Slicing
5.2. Dissolved 10g of ultrafine agarose powder in 500 mL suction-filtered PBS.
SECTION: Preparation for Brain Slicing
5.3. Microwaved until agarose became transparent in the following intervals (30 seconds, 1 minute, 1 minute 30 seconds, 20 seconds).
SECTION: Preparation for Brain Slicing
5.4. Added 500 uL of 20% sodium azide to the agarose mixture. 
SECTION: Preparation for Brain Slicing
5.5. Kept PBS in bead bath at 62°C to maintain in liquid phase.
SECTION: Preparation for Brain Slicing
6. To prepare PBS with 0.02% sodium azide:
SECTION: Preparation for Brain Slicing
5.1. Suction filtered PBS.
SECTION: Preparation for Brain Slicing
6.1. Suction filtered PBS.
SECTION: Preparation for Brain Slicing
6.2. Added 500 uL of 20% sodium azide to 500 mL of suction-filtered PBS.
SECTION: Preparation for Brain Slicing
6.3. Added 1.5 mL of PBS with 0.02% sodium azide to each well of 12-well plate.
SECTION: Brain Slicing
7. Whole brains were embedded in 2% agarose with 0.02% sodium azide by pouring agarose into a mold and letting it set in the 4°C fridge for 10 minutes. 
SECTION: Brain Slicing
8. Irregular borders of the agarose block were cut and removed, and blocks were fixed onto the vibratome stage using Gorilla super glue.
SECTION: Brain Slicing
9. Embedded brains were sliced coronally into 50 µm sections using a vibratome at speed 8 and Personna razor blades. 
SECTION: Brain Slicing
10. Free-floating sections were placed in PBS + 0.02% sodium azide solution and stored at 4°C until staining. 
SECTION: Brain Slicing
10.1. Sections were placed in alternating wells of the 12 well plate. 
SECTION: Immunohistochemistry Preparation
11. Slices containing the striatum were identified and isolated using the Mouse Brain Atlas as a reference.
SECTION: Immunohistochemistry Preparation
12. Slices were permeabilized for 30 minutes at room temperature in 0.5% Triton X-100, 3% BSA in PBS. For each well, 7.5 µL of Triton X-100 and 45 mg of BSA were added to 1.5 mL of filtered PBS. Scaling for 12 wells, 120 µL of Triton X-100 and 720 mg of BSA were added to 24 mL of filtered PBS. 
SECTION: Immunohistochemistry Preparation
13. Slices were washed with submerged for 5 minutes with PBS and washed 3 times. 
SECTION: Immunohistochemistry Preparation
14. Slices were blocked in 10% horse serum in PBS with 0.3% Triton X-100 and 0.04% sodium azide. To make the solution, 5 mL of horse serum was added to 138 µL of Triton X-100, 92 µL of 20% sodium azide, and 45 mL of PBS. 
SECTION: Immunohistochemistry Preparation
15. Slices were washed with submerged for 5 minutes with PBS and washed 3 times. 
SECTION: Immunohistochemistry Preparation
16. Slices were stained overnight at 4˚C while shaking with primary antibody TFAM Rabbit pAb (ABclonal Cat# A1926, RRID:AB_2763953) at a concentration of 1:200 in 0.1% Triton X-100, 3% BSA in PBS. To create the solution, 24 µL of Triton X-100 and 720 mg of BSA were added to 24 mL of PBS. To each well (1.5 mL), 7.5 µL of the primary antibody was added. 
SECTION: Immunohistochemistry Preparation
17. The following day, slices were washed with submerged for 5 minutes with PBS and washed 3 times. 
SECTION: Immunohistochemistry Preparation
18. Slices were stained while shaking with secondary antibody Donkey Anti-Rabbit IgG H&L (Alexa Fluor‱ 647) (Abcam Cat# ab150075, RRID:AB_2752244) at 1:500 for 1 hour and 45 minutes in room temperature. To each well, 3 µL of the secondary antibody was added to 1.5 mL of blocking buffer (PBS with 0.3% Triton X-100 and 0.04% sodium azide). 
SECTION: Immunohistochemistry Preparation
19. Slices were washed with submerged for 5 minutes with PBS and washed 3 times. 
SECTION: Immunohistochemistry Preparation
19.1. In the final wash, 2 µL of DAPI (4′,6-diamidino-2-phenylindole) was added to each well. 


SECTION: Immunohistochemistry Preparation
20. Slices were mounted on a glass cover slip using ProLong Diamond antifade mountant with 4',6-diamidino-2-phenylindole (DAPI). 
SECTION: Imaging and Analysis
21. Cover slips were stored at 4°C until imaging. Imaging was performed on a Zeiss LSM 900 microscope.
SECTION: Imaging and Analysis
22. Images were taken with a 20x objective, with 4-6 pictures taken per animal.
SECTION: Imaging and Analysis
23. ImageJ software was used to measure optical density in striatal region per animal.
SECTION: Imaging and Analysis
24. Values were averaged per animal.