Fluorescence in situ hybridization (FISH)

Detailed Protocol for RNAscope® FISH

The Fluorescence in situ hybridization (FISH) protocol used in Khan et al., 2021* is adapted from the RNAscope® Multiplex Fluorescent Reagent Kit v2 Assay with Sample Preparation and Pretreatment User Manual (Document # 323100-USM). Specifically:

Frozen tissue sample preparation and pretreatment (Pages 19-23)

1. Mouse brains are perfused with freshly prepared 4% paraformaldehyde in 1X PBS and allowed to fix for 24 HR at 4°C.

2. Tissues are immersed in 30% sucrose in 1X PBS with gentle shaking at 4°C until the tissue sank to the bottom of the conical tube.

3. Brains are placed in an embedding mold containing Optimal Cutting Temperature (OCT) embedding media on dry ice. Once the OCT became solid the tissues are stored at -80°C before sectioning.

4. The tissue blocks are allowed to reach to -20°C in a cryostat before sectioning.

5. Sections are cut to a thickness of 25µm and mounted onto SUPERFROST® PLUS SLIDES. 

6. Slides are allowed to air dry for at least 1 HR at -20°C. 

7. Slides are washed with 200 mL 1X PBS in a Tissue-Tek® slide rack for 5 MIN while moving the rack up and down to remove OCT.

8. The slides are baked for 30 MIN at 60°C.

9. Slides are post-fixed by immersing them in prechilled 4% PFA in 1X PBS for 15 MIN at 4°C.

Things to remember: Prepare 200 ml of 50% EtOH, 70% EtOH, and 400 ml 100% EtOH during the 15 MIN incubation period.
 

Tissue Dehydration

10. Slides are removed from 4% PFA then immersed in 50% ethanol (EtOH) for 5 MIN at RT.

11. Slides are removed from 50% EtOH then immersed in 70% EtOH for 5 MIN at RT.

12. Slides are removed from 70% EtOH then immersed in 100% EtOH for 5 MIN at RT.

13. Slides are removed from 100% EtOH then immersed again in 100% EtOH for 5 MIN at RT.

14. Slides are allowed to dry for at least 5 MIN at RT.

RNAscope® Hydrogen Peroxide

15. ~5 drops of RNAscope® Hydrogen Peroxide is added to cover the entire section.

16. Slides are incubated for 10 MIN at RT.

During this incubation: 

17. Prepare 200 mL of 1X RNAscope target retrieval solution from a stock of 20X using distilled water and mix well; initiate warming using a microwave; transfer to a 99°C hot plate.  IMPORTANT:  heat the retrieval solution and additional water within 15 minutes of use.

18. The RNAscope® Hydrogen Peroxide solution is removed from the slide by angling and gently tapping the edge of each slide on absorbent paper. Slides are then submerged in a Tissue-Tek® Staining Dish filled with distilled water.

19. The slides are washed 3-5 times by moving the Tissue-Tek® Slide Rack up and down in the distilled water.

20. Transfer the slide to fresh RT distilled water on an orbital shaker for 2 MIN. 

Target retrieval

21. Slides are first placed in 99°C distilled water for 10 SEC to acclimate, then placed in 1X RNAscope® Target Retrieval Reagent for 15 MIN at 99°C.

22. Slides are removed and placed in a separate container with RT distilled water. Slides are rinsed for 15 SEC.

23. Slides are then transferred to 100% alcohol for 3 MIN.

24. Slides are dried at RT for 5 MIN.

25. Hydrophobic barriers are prepared using an ImmEdge™ hydrophobic barrier pen.

26. The barriers are allowed to dry completely (~ 5 MIN) before proceeding to the next step.

Things to remember: Set the oven to 40°C. Place a humidifying paper in the humidity control tray and use distilled water to wet the paper completely. Cover the tray with a lid and keep it inside the oven. Maintain the tray temperature constant by keeping it inside the oven when not in use.

RNAscope® Protease III

27. Slides are loaded onto the RNAscope EZ-Batch Slide Holder.

28. ~5 drops of Protease III are added to each section.

29. The slide holder is placed into the HybEZ™ Humidity Control Tray and then placed into the HybEZ™ Oven.

30. The samples are incubated for 30 MIN at 40°C.

31. Slides are removed from the oven and washed with 200 mL distilled water using the RNAscope® EZ-Batch™ Wash Tray.

32. Wash step in 31 is repeated with fresh distilled water on an orbital shaker for 2 MIN. 

RNAscope® Multiplex Fluorescent Assay (Pages 28-33)

33. Excess liquid is removed from slides and 4 drops of the probe are added to cover each section. The probe dilution buffer was prepared with 6X saline-sodium citrate buffer (SSC), 0.2% lithium dodecylsulfate (Research Products International; #RPI-L26200), and 20% Calbiochem OmniPur Formamide (#75-12-7).

34. Slides are placed in the tray and inserted into the HybEZ™ Oven for 2 HRS at 40°C.

35. Slides are removed from the oven and washed with 1X Wash Buffer for 2 MIN at RT.

36. Step 35 is repeated with fresh 1X Wash Buffer on an orbital shaker for 2 MIN. 

Things to remember: Prepare 1X Wash Buffer from a 50X stock using distilled water. Warm the stock solution and the probe to 40°C prior to use. Equilibrate AMP 1, AMP 2, AMP 3 and HRP-C1 to RT before use. Ensure the oven is set to 40°C and the humidity control tray is inside the oven.

Hybridize AMP 1

37. Excess liquid is removed from slides then 4 drops of RNAscope® Multiplex FL v2 Amp 1 is added to entirely cover each section.

38. Slides are incubated for 30 MIN at 40°C.

39. Slides are removed from the oven and washed with 1X Wash Buffer for 2 MIN at RT.

40. Wash step is repeated with fresh 1X Wash Buffer on an orbital shaker for 2 MIN. 

Hybridize AMP 2

41. Excess liquid is removed from slides then 4 drops of RNAscope® Multiplex FL v2 Amp 2 is added to entirely cover each section.

42. Slides are incubated for 30 MIN at 40°C.

43. Slides are removed from the oven and washed with 1X Wash Buffer for 2 MIN at RT.

44. Wash step is repeated with fresh 1X Wash Buffer on an orbital shaker for 2 MIN. 

Hybridize AMP 3

45. Excess liquid is removed from slides then 4 drops of RNAscope® Multiplex FL v2 Amp 3 is added to entirely cover each section.

46. Slides are incubated for 15 MIN at 40°C.

47. Slides are removed from the oven and washed with 1X Wash Buffer for 2 MIN at RT.

48. Wash step is repeated with fresh 1X Wash Buffer on an orbital shaker for 2 MIN. 

HRP-C1 signal

49. Excess liquid is removed from each slide then 4 drops of RNAscope® Multiplex FL v2 HRP-C1 is added to entirely cover each section.

50. Slides are incubated for 15 MIN at 40°C.

51. Slides are removed from the oven and washed with 1X Wash Buffer for 2 MIN at RT.

52. Wash step is repeated with fresh 1X Wash Buffer.

53. 50µL of Opal™ 690 (diluted 1:750 in TSA® buffer) is added to each tissue and incubated for 30 MIN at 40°C.

54. Slides are removed from the oven then washed with 1X Wash Buffer for 2 MIN at RT. 

55. Wash step is repeated with fresh 1X Wash Buffer.

56. Excess liquid is removed from the slides then ~4 drops of RNAscope® Multiplex FL v2 HRP blocker is added to entirely cover each section.

57. Slides are incubated into the HybEZ™ Oven for 15 MIN at 40°C.

58. Slides are removed from the oven then washed with 1X Wash Buffer for 2 MIN at RT.

59. Wash step is repeated with fresh 1X Wash Buffer on an orbital shaker for 2 MIN. 

Immunofluorescent Staining and Image Acquisition

60. Slides are incubated with 1X TBS for 5 MIN at RT.

61. Slides are blocked with 0.1% BSA and 10% donkey serum in TBS containing 0.1% Triton X-100 for 30 min. 

62. Slides are washed with blocking buffer without 0.1% Triton X-100 thrice for 5 MIN.

63. Slides are incubated with primary antibodies in TBS containing 1% BSA and 1% DMSO overnight at 4°C. 

64. Slides are washed with blocking buffer without 0.1% Triton X-100 thrice for 5 MIN. 

65. Slides are incubated with secondary antibodies (Donkey-anti Alexa Fluor diluted 1:2000) diluted in TBS containing 1% BSA and 1% DMSO for 2 HRS at RT.

66. Slides are washed with 1X TBS thrice for 5 MIN, then sections are mounted with ProLong Gold Antifade Mountant with DAPI and glass coverslips.

67. Images are captured using a spinning disk confocal microscope (Yokogawa) with an electron multiplying charge coupled device (EMCCD) camera (Andor, UK) and a 100 × 1.4 NA oil immersion objective.

68. ImageJ is used for maximum intensity projections and background subtraction.

69. RNA hybridization is quantified manually for Gli1 and GDNF. Differences in GDNF dots between ciliated and unciliated cells were confirmed using Cell Profiler.

*Reference: 

Khan SS, Sobu Y, Dhekne HS, Tonelli F, Berndsen K, Alessi DR, Pfeffer SR. Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain. Elife. 2021 Oct 18;10:e67900. doi: 10.7554/eLife.67900.