TITLE: Preparation and RNAscope-labeling of fresh mouse midbrain tissue
AUTHORS: William S Conrad

[DESCRIPTION]
Below we describe preparing and labeling coronal sections of mouse substantia nigra (SNc) and ventral tegmental area (VTA) for probes targeting the genes Slc17a6 (VGLUT2), Slc32a1 (VGAT), and Slc18a2 (VMAT2).


[STEPS]
SECTION: A.	Mouse brain extraction
2. Add approximately 50mL of isopentane in a beaker (cleaned with RNAseZap) and chill on dry ice.
SECTION: A.	Mouse brain extraction
3. Anaesthetize mouse with pentobarbital (200 mg/kg i.p.).
SECTION: A.	Mouse brain extraction
4. Decapitate mouse and extract brain from skull.
SECTION: A.	Mouse brain extraction
5. If planning on collecting coronal sections, use a razor blade to block the brain caudal of your region of interest. For example, around -6 mm from bregma for midbrain sections.
SECTION: A.	Mouse brain extraction
6. Immediately snap freeze brain by submerging brain for 30 to 45 seconds in isopentane chilled on dry ice.
SECTION: A.	Mouse brain extraction
7. Place brains in labeled culture tubes pre-chilled on dry ice.
SECTION: A.	Mouse brain extraction
8. Frozen brains can be moved to either an RNAse-free cryostat at −18°C or stored at −80°C.
SECTION: A.	Mouse brain extraction
1. Spray experimenter’s gloves and all tools with RNAseZap before starting and in between brain extractions.
SECTION: B.	Sectioning
9. Set temperature of cryostat objective and chamber to −18°C. 
SECTION: B.	Sectioning
10. Clean interior of cryostat with 100% ethanol. 
SECTION: B.	Sectioning
11. Before sectioning, spray experimenter’s gloves and tools with RNAseZap. Tools that directly touch sections (e.g. brushes, anti-roll plate) should be air dried completely before being placed in cryostat.
SECTION: B.	Sectioning
12. Place culture tube with brain in chamber and let it equilibrate to chamber temperature for about 10 minutes.
SECTION: B.	Sectioning
13. For coronal sections, mount caudal end of brain to chuck using OCT mounting medium. Mount brain as perpendicular to the horizontal plane of the chuck as possible. Equilibrate to chamber temperature again for about 10 minutes.
SECTION: B.	Sectioning
14. Place chuck with brain on objective and adjust orientation to make brain perpendicular to blade. As you begin cutting, adjust brain position so brain landmarks (e.g. corpus collosum and anterior commissure) are as symmetrical as possible. Ensure anti-roll plate is positioned correctly before reaching area of interest.
SECTION: B.	Sectioning
15. Cut coronal brain sections serially at 20 μm on a cryostat throughout the rostro-caudal extent of the region of interest (ROI). For example, SNc and VTA should be collected from -2.5 mm up to and including -4.1 mm from bregma using the Paxinos and Watson atlas (Paxinos & Franklin, 2001).
SECTION: B.	Sectioning
16. Directly mount sections onto Superfrost glass slides. Mount 4 sections per slide.
SECTION: B.	Sectioning
17. Air-dry at room temperature (RT) for 10-20 minutes, then store at -20°C for 2 hours. 
SECTION: B.	Sectioning
18. Slides can be used for RNAscope assay or stored at –80°C in a slide box within an airtight container (e.g. Ziploc bag).
SECTION: C.   DAPI-containing Fluoromount-G preparation
19. Pipet DAPI to Fluoromount-G to reach a concentration of 0.5 µg/mL.
SECTION: C.   DAPI-containing Fluoromount-G preparation
20. Pipet up and down several times to mix, then vortex for at least 30 seconds.
SECTION: C.   DAPI-containing Fluoromount-G preparation
21. Wait at least 24 hours before use. Container should always be covered in foil and stored at 4°C.
SECTION: D.   RNAscope assay
22. Turn on HybEZ™ Oven (ACD) and set to 40°C.
SECTION: D.   RNAscope assay
23. Warm probes in heat-bath for 10 minutes at 40°C, then cool to RT.
SECTION: D.   RNAscope assay
24. Make probe mixture in autoclaved Eppendorf tube by pipetting (with autoclaved pipette tips) 50 parts C1 probe or diluent, 1 part C2 probe (optional) and 1 part C3 probe (optional).  Prepare about 150 µL per slide, assuming each slide contains 4 coronal mouse sections. 
SECTION: D.   RNAscope assay
25. Fix slides in RNAse-free PBS containing 4% paraformaldehyde at 4°C for 15 minutes.
SECTION: D.   RNAscope assay
26. Dehydrate slides in serial ethanol (EtOH) washes: 2 minutes in 50% EtOH, 2 minutes in 70% EtOH, 2 minutes in 100% EtOH twice. Make EtOH dilutions with ultrapure water in sterile conical tubes. 
SECTION: D.   RNAscope assay
27. Air dry slides for 2 to 5 minutes. 
SECTION: D.   RNAscope assay
28. Use hydrophobic pen to draw a barrier around sections. Air dry for 2 to 5 minutes.
SECTION: D.   RNAscope assay
29. Incubate slides with protease IV at RT for 30 min. Keep slides covered to prevent dust contamination.
SECTION: D.   RNAscope assay
30. Rinse slides in RNAse-free 1X PBS at RT.
SECTION: D.   RNAscope assay
31. Decant excess liquid from slides by gently tapping the slide edge on a paper towel. Pipette enough probe mixture to cover sections. 
SECTION: D.   RNAscope assay
32. Incubate for 2 hours in the HybEZ™ Oven at 40°C.
SECTION: D.   RNAscope assay
33. Wash in 1X wash buffer for 2 minutes, twice.
SECTION: D.   RNAscope assay
34. Decant excess liquid from slides and add enough AMP1 to cover sections. Incubate for 30 minutes in the HybEZ™ Oven at 40°C.
SECTION: D.   RNAscope assay
35. Wash in 1X wash buffer for 2 minutes, twice.
SECTION: D.   RNAscope assay
36. Decant excess liquid from slides and add enough AMP2 to cover sections. Incubate for 15 minutes in the HybEZ™ Oven at 40°C.
SECTION: D.   RNAscope assay
37. Wash in 1X wash buffer for 2 minutes, twice.
SECTION: D.   RNAscope assay
38. Decant excess liquid from slides and add enough AMP3 to cover sections. Incubate for 30 minutes in the HybEZ™ Oven at 40°C.
SECTION: D.   RNAscope assay
39. Wash in 1X wash buffer for 2 minutes, twice.
SECTION: D.   RNAscope assay
40. Decant excess liquid from slides and add enough AMP4A to cover sections. Incubate for 15 minutes in the HybEZ™ Oven at 40°C.
SECTION: D.   RNAscope assay
41. Decant excess liquid from slides, add DAPI-containing Fluoromount-G, and coverslip.
SECTION: D.   RNAscope assay
42. Store slides in the dark at 4°C.