TITLE: Combined RNAscope and immunohistochemistry labeling of fresh mouse midbrain tissue
AUTHORS: William S Conrad

[DESCRIPTION]
Here we describe labeling coronal sections of mouse substantia nigra (SNc) and ventral tegmental area
(VTA) for the gene Slc17a6 (VGLUT2) and virally-expressed histone-tagged GFP.

[BEFORE_START]
Tissue should be processed as described previously (dx.doi.org/10.17504/protocols.io.eq2lyjynelx9/v1)

DAPI-containing Fluoromount-G for the final step does not need to be made fresh. However, if it is out of stock it should be made at least one day prior to RNAscope + IHC assay: 
1.    Pipet DAPI to Fluoromount-G to reach a concentration of 0.5 µg/mL.
2.    Pipet up and down several times to mix, then vortex for at least 30 seconds.
3.    Wait at least 24 hours before use. Container should always be covered in foil and stored at 4°C. 

[STEPS]
SECTION: RNAscope assay
1. Turn on HybEZ™ Oven (ACD) and set to 40°C.
SECTION: RNAscope assay
2. Warm probe in heat-bath for 10 minutes at 40°C, then cool to RT.
SECTION: RNAscope assay
3. Drop C1 probe into autoclaved Eppendorf tube. Prepare about 150 µL per slide, assuming each slide contains 4 coronal mouse sections.
SECTION: RNAscope assay
4. Fix slides in RNAse-free PBS containing 4% paraformaldehyde at 4°C for 15 minutes.
SECTION: RNAscope assay
5. 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: RNAscope assay
6. Air dry slides for 2 to 5 minutes.
SECTION: RNAscope assay
7. Use hydrophobic pen to draw a barrier around sections. Air dry for 2 to 5 minutes.
SECTION: RNAscope assay
8. Incubate slides with protease IV at RT for 30 min. Keep slides covered to prevent dust contamination.
SECTION: RNAscope assay
9. Rinse slides in RNAse-free 1X PBS at RT.
SECTION: RNAscope assay
10. Decant excess liquid from slides by gently tapping the slide edge on a paper towel. Pipette enough probe mixture to cover sections.
SECTION: RNAscope assay
11. Incubate for 2 hours in the HybEZ™ Oven at 40°C.
SECTION: RNAscope assay
12. Wash in 1X wash buffer for 2 minutes, twice.
SECTION: RNAscope assay
13. Decant excess liquid from slides and add enough AMP1 to cover sections. Incubate for 30 minutes in the HybEZ™ Oven at 40°C.
SECTION: RNAscope assay
14. Wash in 1X wash buffer for 2 minutes, twice.
SECTION: RNAscope assay
15. Decant excess liquid from slides and add enough AMP2 to cover sections. Incubate for 30 minutes in the HybEZ™ Oven at 40°C.
SECTION: RNAscope assay
16. Wash in 1X wash buffer for 2 minutes, twice.
SECTION: RNAscope assay
17. Decant excess liquid from slides and add enough AMP3 to cover sections. Incubate for 30 minutes in the HybEZ™ Oven at 40°C.
SECTION: RNAscope assay
18. Wash in 1X wash buffer for 2 minutes, twice.
SECTION: RNAscope assay
19. Decant excess liquid from slides and add enough AMP4A to cover sections. Incubate for 30 minutes in the HybEZ™ Oven at 40°C.
SECTION: RNAscope assay
20. Decant excess liquid from slides.
SECTION: RNAscope assay
21. Wash in 1X PBS at RT, twice.
SECTION: Immunohistochemistry (IHC)
22. Incubate each slide with approximately 150 µL of blocking buffer (4% normal donkey serum in 1X PBS with 0.2% Triton X-100) for 1 hour at RT, in the dark.
SECTION: Immunohistochemistry (IHC)
23. Decant excess liquid from slides, then incubate each slide with approximately 150 µL of primary antibody mixture (chicken anti-GFP diluted 1:10,000 in 4% normal donkey serum in 1X PBS with 0.2% Triton X-100) overnight at 4°C, in the dark.
SECTION: Immunohistochemistry (IHC)
24. Wash slides in PBS for 5 minutes at RT, three times.
SECTION: Immunohistochemistry (IHC)
25. Decant excess liquid from slides, then incubate each slide with approximately 150 µL of secondary antibody mixture (donkey anti-Chicken 488 diluted 1:400 in 4% normal donkey serum in 1X PBS) for 2 hours at RT, in the dark.
SECTION: Immunohistochemistry (IHC)
26. Wash slides in PBS for 15 minutes at RT, three times.
SECTION: Immunohistochemistry (IHC)
27. Briefly rinse slides in wash buffer.
SECTION: Immunohistochemistry (IHC)
28. Decant excess liquid from slides, add DAPI-containing Fluoromount-G, and coverslip.
SECTION: Immunohistochemistry (IHC)
29. Store slides in the dark at 4°C.