TITLE: RNAscope for FFPE Mouse Tissue
AUTHORS: Hector Martell Martinez

[DESCRIPTION]
This protocol details the RNAscope for FFPE Mouse Tissue.

[STEPS]
SECTION: DAY 1 - Bake/Adhere
1. Spray RNase away on bench top, slide holder, metal container.
SECTION: DAY 1 - Bake/Adhere
2. Warm oven and slide holder up to 60 °C.

SECTION: DAY 1 - Bake/Adhere
3. Label the lower part of the slide with a pencil.

Can place slides on bench (since sprayed with RNase away).
SECTION: DAY 1 - Bake/Adhere
4. Put slides into slide holder and place into oven → bake for 60 min @ 60 °C.

Meanwhile..
SECTION: DAY 1 - Bake/Adhere
5. Set hyb oven temp to 40 °C.

SECTION: DAY 1 - Bake/Adhere
6. Place slides into tissue tek container and transport to fume hood.
SECTION: DAY 1 - Bake/Adhere
4.1. Turn plate warmer on to 60 °C.
SECTION: DAY 1 - Bake/Adhere
4.2. Make Target Retrieval solution.
SECTION: DAY 1 - Bake/Adhere
4.3. Fill Xylene and Ethanol containers.

 

SECTION: DAY 1 - Bake/Adhere
5.1. Wet humidifying paper with nanopure water (does not need to be dripping).
	
SECTION: DAY 1 - Bake/Adhere
5.2. Place paper on bottom of slide tray and put into the oven for 30 min @ 40 °C.

SECTION: DAY 1 - Deparaffinize
7. 
 
 Xylene container 1 for 5 min.

SECTION: DAY 1 - Deparaffinize
8.  Xylene container 1 for 5 min.
SECTION: DAY 1 - Deparaffinize
7.1. Meanwhile..Turn on vegetable steamer.
SECTION: DAY 1 - Deparaffinize
7.2. When transferring to container 2 → shake off excess.
SECTION: DAY 1 - Deparaffinize
8.1. When transferring to ethanol → shake off excess. 

SECTION: DAY 1 - Deparaffinize
9. Absolute ethanol container 1 for 2 min.
SECTION: DAY 1 - Deparaffinize
9.1. When transferring to container 2 → shake off excess.
SECTION: DAY 1 - Deparaffinize
10. Absolute ethanol container 2 for 2 min.
SECTION: DAY 1 - Deparaffinize
11. Dry on slide warmer for ~5 min @ 60 °C until dry.

SECTION: DAY 1 - Deparaffinize
11.1. Meanwhile... clean bench with RNase away.
SECTION: DAY 1 - Hydrogen Peroxide
12. Place slides on bench top and cover tissue completely with Hydrogen Peroxide (~5-8 drops).

SECTION: DAY 1 - Hydrogen Peroxide
13. Incubate for 10 min @ Room temperature.

Meanwhile..

Boil Target Retrieval solution and a container of Nano pure water in microwave until boiling.

SECTION: DAY 1 - Hydrogen Peroxide
13.1. *Microwave for 1 min at a time*
SECTION: DAY 1 - Hydrogen Peroxide
13.2. When reaches a boil → place containers in warm steamer to ensure solution is at least 99 °C.

SECTION: DAY 1 - Hydrogen Peroxide
14. Remove solution using absorbent paper by tapping long side on paper.

Place in slide holder.
SECTION: DAY 1 - Hydrogen Peroxide
15. Wash slides in slide holder with 200 mL of Nano pure water ~3-5 dunks.

SECTION: DAY 1 - Hydrogen Peroxide
16. Take slides out and repeat step   -  1x.

SECTION: DAY 1 - Target Retrieval Step
17. Keep in water and transport to steamer.
SECTION: DAY 1 - Target Retrieval Step
17.1. Dunk a couple times and soak for 10 s in nanopure water (steamer).

SECTION: DAY 1 - Target Retrieval Step
18. Place in target retrieval solution in the steamer for 30 min (for brain samples) or 15 min (for other tissue).

SECTION: DAY 1 - Target Retrieval Step
19. Rinse slides in fresh 200 mL of Nano pure water → 15 s.

SECTION: DAY 1 - Target Retrieval Step
20. Transfer slides to the 2nd container of absolute ethanol in the fume hood → 3 min.

SECTION: DAY 1 - Target Retrieval Step
21. Dry slides in slide warmer - ~5 min @ 60 °C.

Meanwhile..
SECTION: DAY 1 - Target Retrieval Step
21.1. Rinse slide holder in DI water in sink and let dry on paper towel.
SECTION: DAY 1 - Target Retrieval Step
21.2. Spray bench with RNase away.
SECTION: DAY 1 - Barrier/Protease Plus
22. Put dry slides on bench and square off with hydrophobic pen.
SECTION: DAY 1 - Barrier/Protease Plus
22.1. Leave a little extra room at one side of square to allow space to aspirate later.
SECTION: DAY 1 - Barrier/Protease Plus
23. Apply ~5 drops of protease plus to tissue until sample is completely covered.
SECTION: DAY 1 - Barrier/Protease Plus
24. Incubate in oven for 30 min @ 40 °C.

Meanwhile..
SECTION: DAY 1 - Barrier/Protease Plus
24.1. Warm probes @ 40 °C for ~10 min.

SECTION: DAY 1 - Barrier/Protease Plus
24.2. Create probe solution.

 

SECTION: DAY 1 - Barrier/Protease Plus
25. Wash with DI water in wash tray - 2X.
SECTION: DAY 1 - Barrier/Protease Plus
26. Aspirate with 200 µL pipette tip in the fume hood.
SECTION: DAY 1 - Barrier/Protease Plus
26.1. Wipe bottom of wash tray with big kimwipe.
SECTION: DAY 1 - Hybridize Probe
27. Add ~120 µL of probe mix to respective samples → cover sample completely.
SECTION: DAY 1 - Hybridize Probe
28. Incubate for 120 min @ 40 °C.

SECTION: DAY 1 - Hybridize Probe
29. Wash in 1X wash buffer for 2 minutes 2X.

 

Pour ethanol back into reagent bottle if not doing RNAscope within the next week so doesn't evaporate.
SECTION: DAY 2
30. Spray RNase away on bench top, slide holder, metal container.
SECTION: DAY 2
31. Hydrate Paper and turn on oven to 40 °C.

SECTION: DAY 2
32. Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

SECTION: DAY 2
33. Aspirate.
SECTION: DAY 2
34. Hybridize AMP1.
SECTION: DAY 2
35. Hybridize AMP2.
SECTION: DAY 2
36. Hybridize AMP3.
SECTION: DAY 2
34.1. 4-5 drops covering the sample with AMP1.
SECTION: DAY 2
34.2. Incubate for 30 min @ 40 °C.

SECTION: DAY 2
34.3. Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

SECTION: DAY 2
34.4. Aspirate.
SECTION: DAY 2
35.1. 4-5 drops covering the ample with AMP2.
SECTION: DAY 2
35.2. Incubate for 30 min @ 40 °C.
SECTION: DAY 2
35.3. Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

SECTION: DAY 2
35.4. Aspirate.
SECTION: DAY 2
36.1. 4-5 drops covering the sample with AMP3.
SECTION: DAY 2
36.2. Incubate for 15 min @ 40 °C.
SECTION: DAY 2
36.3. Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

SECTION: DAY 2
36.4. Aspirate.
SECTION: Develop Channels (C1-C3)
37. 
 
Develop HRP-C2 (570 or 690) signal.

SECTION: Develop Channels (C1-C3)
38. Repeat   using HRP-C3.

SECTION: Develop Channels (C1-C3)
39. For 780 Channel
SECTION: Develop Channels (C1-C3)
37.1. Add 4-5 drops covering the sample with HRP-C2 (or respective HRP-C#) and incubate for 15 min @ 40 °C.

Meanwhile.. Dilute Opal dyes → KEEP IN DARK 
                          1 µL Dye (570 or 690) + 1000 µL TSA buffer

SECTION: Develop Channels (C1-C3)
37.2. Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

Aspirate.
SECTION: Develop Channels (C1-C3)
37.3. Add 1st Opal dye and Incubate for 30 min @ 40 °C.
SECTION: Develop Channels (C1-C3)
37.4. Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

Aspirate. 
SECTION: Develop Channels (C1-C3)
37.5. Add 4-6 drops HRP blocker and incubate for 15 min @ 40 °C.
SECTION: Develop Channels (C1-C3)
37.6. Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

Aspirate. 
SECTION: Develop Channels (C1-C3)
39.1. Add HRP-C1 and incubate for 15 min @ 40 °C.

Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

Aspirate. 
SECTION: Develop Channels (C1-C3)
39.2. Add ~120 µL of TSA-DIG per section and incubate for 30 min @ 40 Room temperature.

Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

Aspirate. 
SECTION: Develop Channels (C1-C3)
39.3. Add HRP Blocker and incubate for 15 min @ 40 °C.

Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

Aspirate. 
SECTION: Develop Channels (C1-C3)
39.4. Add ~120 µL of Opal 780 per section and incubate for 30 min @ 40 Room temperature.

Wash slides with 1X wash buffer for 2 minutes with slight agitation 2X.

Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).

Aspirate. 
SECTION: Quench w/ TrueBlack Plus
40. Wash w/ PBS 3X.

Aspirate.
SECTION: Quench w/ TrueBlack Plus
41. Apply ~120 µL of 1X TrueBlack Plus for 10 min @ Room temperature.

SECTION: Quench w/ TrueBlack Plus
42. Wash w/ PBS 3X.

Aspirate.
SECTION: DAY 1 - Hybridize Probe
29.1. Wash in 1X wash buffer for 2 min (1/2).
SECTION: DAY 1 - Hybridize Probe
29.2. Wash in 1X wash buffer for 2 min (2/2).
SECTION: DAY 2
32.1. Wash slides with 1X wash buffer for 2 min with slight agitation (1/2).
SECTION: DAY 2
32.2. Wash slides with 1X wash buffer for 2 min with slight agitation (2/2).
SECTION: DAPI and Mount
43. Add 4-5 drops of DAPI to completely cover section.
SECTION: DAPI and Mount
44. Incubate for 30 s @ Room temperature.

SECTION: DAPI and Mount
45. Remove DAPI with absorbent paper → tap long side gently to paper.
SECTION: DAPI and Mount
46. Add 3-4 drops of Prolong Gold Mountant.
SECTION: DAPI and Mount
47. Add Coverslip.
SECTION: DAPI and Mount
47.1. Use a forceps to attach coverslip.
SECTION: DAPI and Mount
47.2. Gently press down.
SECTION: DAPI and Mount
47.3. Kim wipe the bottom of the slide and the bench in between samples.
SECTION: DAPI and Mount
48. Store in dark 30 s on absorbent paper (in a drawer).

Dry slides in the dark overnight and then Store slides at 4 °C the next day.