TITLE: Multicolor fluorescence in situ hybridization and analysis
AUTHORS: Chuyu Chen

[DESCRIPTION]
Antipsychotics are known to induce the expression of several immediate early genes (IEGs) via D2R antagonism, which then leads to changes in critical signaling pathways in iSPNs. To define the impact of LRRK2 kinase activity on the expression pattern of IEGs in the iSPNs, we used multicolored single-molecule fluorescence in situ hybridization.

[STEPS]
SECTION: Fresh Frozen Tissue Prep
1. Place a piece of aluminum foil into a small Styrofoam container containing dry ice.
SECTION: Fresh Frozen Tissue Prep
2. Mice were euthanized with carbon dioxide, decapitated, their brains rapidly removed, and placed immediately on foil on dry ice
SECTION: Fresh Frozen Tissue Prep
3. Pour ~50 mL of 2-methylbutance into the container. Keep brain in container for 2 minutes.
SECTION: Fresh Frozen Tissue Prep
4. When the brain is completely frozen, carefully remove it from the foil with forceps and place it in
the OCT mold. 
SECTION: Fresh Frozen Tissue Prep
5. Pour ~50 mL of 2-methylbutance into the container. Keep brain in container for 3 minutes.
SECTION: Fresh Frozen Tissue Prep
6. Cover the top of the mold with tin foil and store the brain in -80°C freezer. Place the brain into -20°C freezer 20 mins before cryosection.
SECTION: Fresh Frozen Tissue Prep
7. collect the region of interest with 20μm slices and mount by turning the slide upside down and pressing against the slice on theplatform.
SECTION: Fresh Frozen Tissue Prep
8. Place slides in slide box and store slides in -80°C freezer.
SECTION: Fix and Dehydrate slides
9. Prepare 200 mL of 4% PFA and chilled to 4°C:
SECTION: Pretreat samples with Hydrogen Peroxide and Protease
18. Load the slides into the EZ-Batch holder, and add 5 drops of RNAscope Hydrogen Peroxide to each section (completely cover the sections). incubate at RT for 10 minutes.
SECTION: Fix and Dehydrate slides
10. Place slides in the staining tank containing chilled 4% PFA in fridge (4°C) for 30mins
SECTION: Fix and Dehydrate slides
11. Wash slides in 1X PBS by moving the rack gently up and down for 2 minutes and repeat with fresh 1X PBS (room temperature)
SECTION: Fix and Dehydrate slides
12. Place slide rack in new staining tank containing 200 mL of 50% EtOH for 5 minutes at room temperature. Gently agitate slides by moving rack up and down 2-4 times
SECTION: Fix and Dehydrate slides
13. Place slide rack in new staining tank containing 200 mL of 70% EtOH for 5 minutes at room temperature. Gently agitate slides by moving rack up and down 2-4 times.     
SECTION: Fix and Dehydrate slides
15. Place slide rack in new staining tank containing 200 mL of 100% EtOH for 5 minutes at room temperature. Gently agitate slides by moving rack up and down 2-4 times. 
SECTION: Fix and Dehydrate slides
14. Place slide rack in new staining tank containing 200 mL of 100% EtOH for 5 minutes at room temperature. Gently agitate slides by moving rack up and down 2-4 times. 
SECTION: Fix and Dehydrate slides
13.1. Turn on the HbyEZ oven and prepare the humidity contral tray by applying ~40 mL of MilliQ water to paper towels.
SECTION: Fix and Dehydrate slides
14.1. Allow Protease IV to come to RT (normally stored at 4°C)
SECTION: Fix and Dehydrate slides
15.1. Warm probes for 10-15 minutes at 40°C
SECTION: Fix and Dehydrate slides
16. Practice making hydrophobic barriers using ImmEdge pen
SECTION: Fix and Dehydrate slides
17. Remove probes from oven, allow to come to RT
SECTION: Pretreat samples with Hydrogen Peroxide and Protease
19. Place the EZ-Batch holder into the wash tray containing 200ml distilled water.  
SECTION: Pretreat samples with Hydrogen Peroxide and Protease
20. Repeat the wash step with fresh water.
SECTION: Pretreat samples with Hydrogen Peroxide and Protease
21. Add 5 drops of RNAscope Protease IV to each section (completely cover the sections). incubate at RT for 30 minutes.
SECTION: Pretreat samples with Hydrogen Peroxide and Protease
22. Place the EZ-Batch holder into the wash tray containing 200ml PBS
SECTION: Pretreat samples with Hydrogen Peroxide and Protease
23. Repeat the wash step with fresh PBS
SECTION: Multicolor fluorescence in situ hybridization
25. Preparing probes by ratio 50:1:1 for channels 1, 2, and 3
SECTION: Pretreat samples with Hydrogen Peroxide and Protease
24. Allow slides to dry. Collect excess PBS with Kimwipe
SECTION: Multicolor fluorescence in situ hybridization
26. Add mixed probes to each section. Apply ~100 μL of probe combination to each section (completely cover the sections) and incubate at 40°C  for 2hrs.
SECTION: Multicolor fluorescence in situ hybridization
35. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
34. Apply ~3 drops of Amp 2 to each section(completely cover the sections) and incubate at 40°C for 30 minutes
SECTION: Multicolor fluorescence in situ hybridization
33. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
32. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
31. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
30. Apply ~3 drops of Amp 1 to each section(completely cover the sections) and incubate at 40°C for 30 minutes.
SECTION: Multicolor fluorescence in situ hybridization
29. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
28.  Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
27. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
26.1. 30 minutes before the end of incubation: remove amps from the fridge and allow amps to come to RT
SECTION: Multicolor fluorescence in situ hybridization
40. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
39. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
38. Apply ~3 drops of Amp 3 to each section(completely cover the sections) and incubate at 40°C for 15 minutes
SECTION: Multicolor fluorescence in situ hybridization
37. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
36. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
52. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
51. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
50. Apply ~3 drops of HRP blocker to each section(completely cover the sections) and incubate at 40°C for 15 minutes
SECTION: Multicolor fluorescence in situ hybridization
49. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
48. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
47. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
46. Apply 150μl of Opal 520 to each section(completely cover the sections) and incubate at 40°C for 30 minutes
SECTION: Multicolor fluorescence in situ hybridization
45. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
44. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
43. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
42. Apply ~3 drops of HRP-C1 to each section(completely cover the sections) and incubate at 40°C for 15 minutes
SECTION: Multicolor fluorescence in situ hybridization
41. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
63. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
62. Apply ~3 drops of HRP blocker to each section(completely cover the sections) and incubate at 40°C for 15 minutes
SECTION: Multicolor fluorescence in situ hybridization
61. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
60. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
59. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
58. Apply 150μl of Opal 570 to each section(completely cover the sections) and incubate at 40°C for 30 minutes
SECTION: Multicolor fluorescence in situ hybridization
57. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
56. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
55. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
54. Apply ~3 drops of HRP-C2 to each section(completely cover the sections) and incubate at 40°C for 15 minutes
SECTION: Multicolor fluorescence in situ hybridization
53. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
78. Apply ~3 drops of DAPI to each section(completely cover the sections) and incubate at RT for 1 minutes
SECTION: Multicolor fluorescence in situ hybridization
70. Apply 150μl of Opal 650 to each section(completely cover the sections) and incubate at 40°C for 30 minutes
SECTION: Multicolor fluorescence in situ hybridization
66. Apply ~3 drops of HRP-C3 to each section(completely cover the sections) and incubate at 40°C for 15 minutes
SECTION: Multicolor fluorescence in situ hybridization
65. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
64. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
67. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
68. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
69. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
71. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
72. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
73. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
75. Flick off excess liquid and wash slides in 1X Wash Buffer (200ml) for 2 minutes
SECTION: Multicolor fluorescence in situ hybridization
76. Repeat with fresh 1X Wash Buffer in fresh wash buffer
SECTION: Multicolor fluorescence in situ hybridization
77. Remove excess liquid
SECTION: Multicolor fluorescence in situ hybridization
74. Apply ~3 drops of HRP blocker to each section(completely cover the sections) and incubate at 40°C for 15 minutes
SECTION: Multicolor fluorescence in situ hybridization
79. Flick off excess liquid and mount the slides with Prolong Diamond Antifade mountant.

SECTION: Multicolor fluorescence in situ hybridization
80. Allow slides to dry in protected and covered area overnight. store slides in the dark at 4°C
SECTION: Confocal images capture
81. Sections were imaged with the Nikon A1 laser microscope system using a 60X 0.75NA objective, to capture 3 z-stack images across 4 channels:
SECTION: Image analysis with Cell Profiler
82. DAPI channel was enhanced with Enhance Or Suppress Features module, with feature size set at pixel size 30.
SECTION: Image analysis with Cell Profiler
87.  Nur77 or Arc object pixel unit was set for Min 1, Max10, threshold strategy for Global and thresholding method set as Manual
SECTION: Image analysis with Cell Profiler
84. Drd1 or Drd2 channel was enhanced with Enhance Or Suppress Features module, with feature size set at pixel size 10
SECTION: Image analysis with Cell Profiler
83. Identify Primary Objects module was implemented on identified nuclei, object pixel unit was set for Min 5, Max 50, with threshold strategy set as Global and thresholding method set as Otsu
SECTION: Image analysis with Cell Profiler
88. Masked D1 positive nuclei and Masked D2 positive nuclei were generated with RelateObjects
SECTION: Image analysis with Cell Profiler
86. Identify Primary Objects module was implemented on identified Drd1 or Drd2 signal puncta, object pixel unit was set for Min 3, Max30, threshold strategy for Global and thresholding method set as Manual
SECTION: Image analysis with Cell Profiler
85. Nr4a1 or Arc channel was enhanced with Enhance Or Suppress Features module, with feature size set at pixel size 5.
SECTION: Image analysis with Cell Profiler
89.  The number of objects were measured with the Measure Object Intensity module.