TITLE: Dual In Situ Hybridization/Immunofluorescence
AUTHORS: Michael Henderson

[DESCRIPTION]
This protocol details about the Dual In Situ Hybridization/Immunofluorescence in tissue.

[GUIDELINES]
ACD protocol notes that tissues should be fixed in 10% NBF for 16-32 hours, and embedded in  paraffin. Then, sectioned and dried overnight at RT. They suggest sectioned tissue be used in less  than a year (4°C) or less than 3 months at room temperature.

[STEPS]
SECTION: Preparing Tissue (Day 1): Prepare Tissue
1. Bake slides in a dry oven for 60 min at 60 °C. Use slides within a week.

SECTION: Preparing Tissue (Day 1): Prepare Tissue
2. De-paraffinize slides in fresh xylenes, then in 100% ethanol.
SECTION: Preparing Tissue (Day 1): Prepare Tissue
3. Place slides on absorbent paper and dry in the oven from5 min at 60 °C or until dry.

SECTION: Preparing Tissue (Day 1): Prepare Tissue
2.1. De-paraffinize slides for  5 min in fresh xylenes. (1/4)

SECTION: Preparing Tissue (Day 1): Prepare Tissue
2.2. De-paraffinize slides for 5 min in fresh xylenes. (2/4)
SECTION: Preparing Tissue (Day 1): Prepare Tissue
2.3. De-paraffinize slides for 2 minin 100% ethanol. (3/4)

SECTION: Preparing Tissue (Day 1): Prepare Tissue
2.4. De-paraffinize slides for 2 minin 100% ethanol. (4/4)
SECTION: Preparing Tissue (Day 1): Hydrogen Peroxide Treatment
4. Place slide horizontally in an incubation tray. Add ~5-8 drops of RNAscope Hydrogen Peroxide to cover each section. Incubate for 10 min at 60 Room temperature.

SECTION: Preparing Tissue (Day 1): Hydrogen Peroxide Treatment
5. Dab solution off and move to a rack in distilled water. Move up and down 5 times. Repeat with a fresh boat of distilled water.
SECTION: Preparing Tissue (Day 1): Target Retrieval
6. Dilute Target Retrieval Regents (RNAscope) 1:10 in dH2O (25 mL/225 mL dH2O/boat). Mix well.

SECTION: Preparing Tissue (Day 1): Target Retrieval
7. Place in microwave for 15 minat 95 °C.

SECTION: Preparing Tissue (Day 1): Target Retrieval
8. Transfer slides to a slide boat with 200 mL distilled water for 15 s.

SECTION: Preparing Tissue (Day 1): Target Retrieval
9. Transfer the slides to 100% ethanol for 3 min.

SECTION: Preparing Tissue (Day 1): Target Retrieval
10. Dry the slides in a 60 °C incubator (or 60 Room temperature) for 5 min.

SECTION: Preparing Tissue (Day 1): Target Retrieval
11. Draw a hydrophobic barrier onto slides with ImmEdge pen. Do NOT due for fluorescent slides. Let the barrier dry for5 min. OPTIONAL PAUSE POINT 5 min at60 Room temperature.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Protease Treatment
12. Place a wet Humidifying Paper in an incubation tray and warm for 30 min at 40 °C(TC incubator). Keep the tray in the incubator when not in use. Insert the slides into the incubation tray.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Protease Treatment
13. Add ~5 drops RNAScope Protease Plus (Protease III-Cheadle) to cover each section and place tray into the incubator at40 °C for30 min (standard) (15 min-Otero-Garcia). 
 

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Protease Treatment
14. Wash slides with 200 mL+ distilled water and slight agitation.
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Protease Treatment
14.1. Wash slides with 200 mL+ distilled water and slight agitation. (1/2)

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Protease Treatment
14.2. Wash slides with 200 mL+ distilled water and slight agitation. (2/2)

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Preparation
15. Wash Buffer: Warm 50x Buffer to 40 °C for 10 min to20 min. Add 980 mL distilled water to 20 mL of RNAscope Wash Buffer in a 1 Lbottle. May need 1 L to 2 L per run. Mix well. Can be stored for up to one month.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Preparation
16. Probes: Prepare only those probes needed. 
 
Warm probes for 10 min at 40 °C, then let cool to40 Room temperature. Add 1 volume C2 and volume C3 probes to 50 volumes C1 probe in a tube (e.g. 200 µL C1 + 4 µL C2). Invert to mix. Store at 4 °C for up to 6 months.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Preparation
17. Reagents: Warm AMP1-3, HRP-C1-3 and HRP blocks at 4 Room temperature.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Preparation
18. (Optional) Saline Sodium Citrate: 175.3 g NaCl + 88.2 g sodium citrate in 800 mListilled water. Adjust topH 7.0 with 1 Molarity (M) HCl. Add water to a final volume of 1 L. Sterilize by autoclaving and store at 4 Room temperature for up to 2 months.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize Probes
19. Remove liquid from slides. Add 4-6 drops (6 drops=180 µL) of the probe mix to slides. Incubate in incubator for 120 min at 40 °C.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize Probes
20. Wash slides with Wash Buffer.
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize Probes
21. OPTIONAL PAUSE POINT: Store slides in 5x SSC 2 min at 40 Room temperature.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize Probes
20.1. Wash slides with Wash Buffer 2 min at 40 Room temperature. (1/2)

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize Probes
20.2. Wash slides with Wash Buffer 2 min at 40 Room temperature. (2/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize AMPs
22. Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 Amp 1 to each slide. Incubate in incubator for 30 min at40 °C.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize AMPs
23. Wash slides with Wash Buffer.
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize AMPs
24. Repeat steps 22 and 23 for Amp 2 and Amp 3. Amp 3 only requires 15 min at 40 °C.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize AMPs
25. During this incubation, dilute necessary Opal Dye fluorophores in TSA Buffer (1:1500 standard).

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize AMPs
23.1. Wash slides with Wash Buffer 2 min at 40 Room temperature. (1/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Hybridize AMPs
23.2. Wash slides with Wash Buffer 2 min at 40 Room temperature. (2/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
26. Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 HRP-C1 to each slide. Incubate in incubator for 15 min at 40 °C.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
27. Wash slides with Wash Buffer.
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
28. Remove liquid from slides. Add 200 µL Opal 520 to each slide. Incubate in HybEZ Oven for30 minat 40 °C.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
29. Wash slides with Wash Buffer.
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
30. Remove liquid from slides. Add 4-6 drops RNAScope Multiplex FL v2 HRP Blocker to each slide. Incubate in incubator for 15 min at 40 °C.

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
31. Wash slides with Wash Buffer.
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
32. STOP HERE IF USING JUST C1 PROBE. Continue to Immunofluorescence.
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
33. Repeat steps 26-32 with HRP-C2 and Opal 570, and again with HRP-C3 and Opal 690. 
 

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
27.1. Wash slides with Wash Buffer 2 min at 40 Room temperature. (1/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
27.2. Wash slides with Wash Buffer 2 min at 40 Room temperature. (2/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
29.1. Wash slides with Wash Buffer 2 min at 40 Room temperature. (1/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
29.2. Wash slides with Wash Buffer 2 min at 40 Room temperature. (2/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
31.1. Wash slides with Wash Buffer 2 min at 40 Room temperature. (1/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
31.2. Wash slides with Wash Buffer 2 min at 40 Room temperature. (2/2)
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
33.1.  4% PFA fix for 15 min at 4 °C. 
SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
33.2. Then,  wash with PBS-Otero-Garcia for 4 min. (1/2)

SECTION: RNAscope Multiplex Fluorescent v2 Assay (Day 2): Develop HRP Signals
33.3.  Wash with PBS-Otero-Garcia for 4 min. (2/2)
SECTION: Day 2: Immunofluorescence
34. Wash in0.1 Molarity (M) Tris buffer, pH 7.6 5 min. Discard all Tris washes.

SECTION: Day 2: Immunofluorescence
35. Block in 0.1 Molarity (M) Tris/2% FBS (Tris/FBS) 30 min+. Keep blocking solution for up to 2 weeks @ 4 °C.

SECTION: Day 2: Immunofluorescence
36. Dilute primary antibodies in Tris/FBS), and prepare humidified chamber(s) by soaking towel in the middle of the slide chamber(s).
SECTION: Day 2: Immunofluorescence
37. Wipe excess fluid off back of slides and from around tissue and apply200 µL of primary antibody to slides.

SECTION: Day 2: Immunofluorescence
38. Incubate at4 °C in humidified chamber45 min to 120 min at 4 Room temperatureor 120 min at 4 °C.

SECTION: Day 3
39. Rinse off antibody from tissue using Tris. 
 

SECTION: Day 3
40. Wash in Tris 5 min.

SECTION: Day 3
41. Block in Tris/FBS 5 min.

SECTION: Day 3
42. Dilute fluorophore-conjugated secondary antibody 1:500 in Tris/FBS and apply 200 µL to wiped slides. Incubate at4 Room temperature for 120 min or 120 minat 4 °C.

SECTION: Day 3
43. Rinse off slides with Tris.
SECTION: Day 3
44. Wash in running tap H2O for 5 min.

SECTION: Day 3
45. Wash in Tris for 5 min in green boats.

SECTION: Day 3
46. Coverslip using non-photobleaching reagent (Prolong Gold with DAPI or FluorMount with DAPI). Allow to dry completely before imaging on scanner.