TITLE: RNAScope in situ hybridization (ISH) multiplex fluorescent protocol
AUTHORS: Roberta Marongiu, Santiago Unda

[DESCRIPTION]
Instructions to perform ISH using RNAscope kit from acdbio company 

[BEFORE_START]
For Fixed Frozen Brain samples 

The challenge with thicker sections (30 to 40 microns) is tissue attachment. Hence, there are additional fixation and baking steps with respect to the standard protocol.

- Cut 30 μm (or 40 μm) sections. 
- Collect sections into 12-24 well plates in anti-freeze solution
- Store sections at -20oC for no more than 3-4 weeks; at 4°C for no more than 2 days.

For Fixed Frozen Nodose Ganglion samples 

- Cut 14 to 20 μm sections in cryostat. 
- Collect sections into SuperFrost Plus Gold slides, avoid more than 3 sections per slide, and OCT overlapping with tissue from a previous section.
- Let sections dry at room temperature for 1 hour.
- Store sections at -80oC for no more than 4-5 weeks.



[GUIDELINES]
ISH is used to detect RNA. Thus, it is very important that all the reagents, equipment, and bench space used are very clean and uncontaminated.





[STEPS]
SECTION: Part 1 : Sample preparation for frozen Nodose ganglia samples
1. Select tissue – ~ 3 sections/sample.
SECTION: Part 1 : Sample preparation for frozen Nodose ganglia samples
2. Dry tissue at Room temperature for 60 min to 120 min.  
Cover the slide loosely with foil to avoid contaminations.
SECTION: Part 1 : Sample preparation for frozen brain samples
4. Select tissue – ~ 3 sections/sample.
SECTION: Part 1 : Sample preparation for frozen brain samples
5. Wash in PBS 1X to remove agar or OCT for 2X 15 min. 
Wash free-floating sections into net-wells and black tray.
SECTION: Part 1 : Sample preparation for frozen brain samples
7. Dry at Room temperature for 60 min - cover the slides loosely with foil to avoid contaminations.
SECTION: Part 1 : Sample preparation for frozen brain samples
8. Rinse in ddH2O by moving slide rack up and down (3-5 times) for 3x 15 s.

SECTION: Part 1 : Sample preparation
9. Dry at Room temperature for 60 min - cover the slides loosely with foil to avoid contaminations.
SECTION: Part 1 : Sample preparation
11. Bake at 60 °C for 60 min.
SECTION: Part 1 : Sample preparation
12. During this incubation, prepare 4% PFA and keep in the fridge 4 °C .
SECTION: Part 1 : Sample preparation
13. Post-fix in 4% PFA at 4 °C for 60 min (in the fridge).

SECTION: Part 1 : Sample preparation
14. During this time prepare the following buffers:

SECTION: Part 1 : Sample preparation
14.1. Make 1X target retrieval buffer   .
(Warm stock solution 10X in water bath at 40°C for ~10 min before diluting / 1 mL 10X stock solution + 9 mL ddH20 )
SECTION: Part 1 : Sample preparation
14.2. Make 1X wash buffer ( provided in  ). 
- Warm 50X stock solution in water bath at 40°C for 10-20 min before diluting 
- 1mL 50X stock solution + 49 mL ddH20. MIX WELL!!!
- 3L of 1X wash buffer -> 60 mL 50x stock solution (1 bottle) + 2.94 L ddH20
- Store 1X wash buffer at RT for up to 1 month.
SECTION: Part 1 : Sample preparation
14.3. Prepare bottles with EtOH gradient solutions: 50%, 70%, and 100%.
SECTION: Part 2: RNAscope pre-treatments
16. TURN ON STEAMER and SETUP at 100 °C. Use thermometer to check temperature.
SECTION: Part 2: RNAscope pre-treatments
17. Dry at 60 °C for 15 min.
SECTION: Part 2: RNAscope pre-treatments
18. Add RNAscope hydrogen peroxide  - incubate slides at 100 Room temperature for 10 min.
SECTION: Part 2: RNAscope pre-treatments
19. If active bubbling happens, please extend the 100 Room temperature incubation to 20 min.
SECTION: Part 2: RNAscope pre-treatments
20. 15.  Rinse in ddH2O by moving slide rack up and down (3-5 times) for 2 x 15 s.
SECTION: Part 2: RNAscope pre-treatments
21. Dry at 60 °C for 15 min.
SECTION: Part 2: RNAscope pre-treatments
22. PLACE JARS WITH ddH2O AND TARGET RETRIEVAL (TR) SOLUTION INTO STEAMER 15 minbefore is needed.  Before starting the target retrieval step make sure that the TR buffer is not actively bubbling.
SECTION: Part 2: RNAscope pre-treatments
23. TR step in the steamer -> place slides in ddH2O jar for 15 sec then move them into TR solution jar for 10 min100 °C.
SECTION: Part 2: RNAscope pre-treatments
24. Rinse in ddH2O 15 s by moving slide rack up and down (3-5 times).

SECTION: Part 2: RNAscope pre-treatments
26. Dry 60 °C for 10 min.

SECTION: Part 2: RNAscope pre-treatments
30. Rinse in ddH2O by moving slide rack up and down (3-5 times) for 2 x 15 s.
SECTION: Part 2: RNAscope pre-treatments
31. After this step, proceed IMMEDIATELLY to the RNAscope assay.
SECTION: Part 2: RNAscope pre-treatments
15. *NOTE – After each step (most important after washes)- Remove excess liquid from the slides before adding new reagents by tapping or gently flicking the slide on absorbent paper.
If you have a hydrophobic barrier, you can also use a Kimwipe to absorb the left-over liquid. Tilting the slide slightly and allowing solutions to pool in one corner on the hydrophobic barrier to then dab with a Kimwipe works well.

Dehydration steps using EtOH gradient: 50%, 70%, 100%, new 100% - at Room temperature, 5 min/each.
SECTION: Part 3: RNAscope assay
32. PROBE HYBRIDIZATION
Add (4-6) drops of probe mixture to appropriate slide(s), add positive probe to positive control slide, add negative probe to negative control slide. Incubate at 40 °C for 120 min.

SECTION: Part 3: RNAscope assay
33. During this time prepare 5x SSC buffer: 
1.25mL 20x SSC + 3.75mL ddH20
5x SSC buffer can be stored for up to 2 months at RT   
SECTION: Part 3: RNAscope assay
34. Rinse with 1X wash buffer for 2x 2 min.
SECTION: Part 3: RNAscope assay
35. Cover tissue with 5x SSC, incubate at 8 Room temperature2 min in covered humidified chamber.

SECTION: Part 3: RNAscope assay
36. DAY 2

TURN ON HYBRIDIZATION OVEN and setup it at 40 °C.
SECTION: Part 3: RNAscope assay
37. Remove 5x SSC solution.
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38. Rinse with 1X wash buffer at8 Room temperature for 2 min.
SECTION: Part 3: RNAscope assay
39. Add drops of Amp #1 at 40 °C for 30 min(in humidified chamber into hybridization oven).
SECTION: Part 3: RNAscope assay
40. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
41. Add drops of Amp #2 at 40 °C for 330 min.
SECTION: Part 3: RNAscope assay
42. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
43. Add drops of Amp #3 at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
44. During this time, dilute Opal fluorophores (range 1:750-1:3K; usually dil. 1:1500) in TSA buffer:
SECTION: Part 3: RNAscope assay
44.1. Determine volume needed (150-220 ul/slide).
Example: 0.5μL fluorophore in 750μL TSA buffer from kit
SECTION: Part 3: RNAscope assay
44.2. Use tinfoil to cover tubes with the solutions as fluorophores are light sensitive.

NOTE: if Opal 690 is assigned to C1 or C2, its concentration may need to be increased.
SECTION: Part 3: RNAscope assay
45. Rinse with 1X wash buffer at8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
46. Add drops of HRP-C1 at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
47. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
48. Add drops of diluted Opal selected for C1 at 40 °C for 30 min.
SECTION: Part 3: RNAscope assay
49. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
50. Add drops of HRP-blocker  at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
51. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
52. Add drops of HRP-C2 at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
53. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
54. Add drops of diluted Opal selected for C2 at 40 °C for 30 min.
SECTION: Part 3: RNAscope assay
55. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
56. Add drops of HRP-blocker  at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
57. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
58. Add drops of HRP-C3 at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
59. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
60. Add drops of diluted Opal selected for C3 at 40 °C for 30 min.
SECTION: Part 3: RNAscope assay
61. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
62. Add drops of HRP-blocker  at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
63. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
64. Add drops of HRP-C4  at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
65. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
66. Add drops of diluted Opal selected for C4 at at 40 °C for 30 min.
SECTION: Part 3: RNAscope assay
67. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
68. Add drops of HRP-blocker  at 40 °C for 15 min.
SECTION: Part 3: RNAscope assay
69. Rinse with 1X wash buffer at 8 Room temperature for 2x 2 min.
SECTION: Part 3: RNAscope assay
70. DAPI solution from RNAscope kit at 8 Room temperature for 30 s.

SECTION: Part 3: RNAscope assay
71. Air dry slides briefly, covered (slides are light sensitive).
SECTION: Part 3: RNAscope assay
72. Coverslip with ProLong Gold Antifade Mountant (from Thermo Fisher) or other comparable fluorescent mountant.
SECTION: Part 3: RNAscope assay
73. Immediately store slides in a light-proof box at 4 °C Dry 30 min to O/N.

SECTION: Part 3: RNAscope assay
73.1. Do not leave out at RT.

SECTION: Part 3: RNAscope assay
73.2. To prolong the signal, clear nail polish may be used to seal the coverslip to the slide.

SECTION: Part 3: RNAscope assay
73.3. Image slides after 8 hours or within 2 weeks. Signal deteriorates fast.
SECTION: Part 2: RNAscope pre-treatments
25. Dip in 100% EtOH at Room temperature for 3 min.

SECTION: Part 2: RNAscope pre-treatments
27. Draw hydrophobic barrier around the tissue:
Using the hydrophobic pen,  , make a barrier as close to tissue as possible (without touching it) to reduce the amount of solution needed to cover the surface area. 

While waiting for pen to dry, PRE-HEAT OVEN and SLIDE TRAY AT 40 °C.
SECTION: Part 2: RNAscope pre-treatments
28. Add (4-6) drops of protease III (provided in    )  , digest in humidity chamber at 40 °C for 30 min .
SECTION: Part 2: RNAscope pre-treatments
29. During protease incubation:
SECTION: Part 2: RNAscope pre-treatments
29.1. Equilibrate amplification reagents (provided in  ) :
    Remove AMP1, AMP2, AMP3, HPR-C1, HPR-C2, HPR-C3, HPR blockers from the fridge and equilibrate at RT.
SECTION: Part 2: RNAscope pre-treatments
29.2. Prepare the probes (provided in   )
    Warm probes and probe stock solution in water bath at 40 °C for 10 min
    cool at 40 Room temperature
    Briefly spin probe stock solutions
    Mix probes into a new tube - Probe ratios = C2:C3:C4:C1 = 1:1:1:50. 
            Example: 4μL C3 + 4μL C2 + 192 μL C1
            If not using a C1 probe, use RNAScope probe diluent in its place
    Invert tube several times to mix before use.
    Store mixed probes at 2 °C-8 °C for up to 6 months.
SECTION: Part 1 : Sample preparation for frozen Nodose ganglia samples
3. Wash in PBS 1X to remove OCT for 2X 15 min.

SECTION: Part 1 : Sample preparation for frozen brain samples
6. Mount tissue on superfrost charged gold slides.
SECTION: Part 1 : Sample preparation
10. During this incubation, PRE-HEAT HYBRIDIZATION OVEN and SLIDE TRAY at 60 °C.