TITLE: Targeted detection of SNCA CNVs in SOX10+ nuclei from oligodendrocytes  containing alpha-synuclein inclusions isolated from human post-mortem brain
AUTHORS: Caoimhe Morley, Diego-Perez Rodriguez, Monica Emili Garcia-Segura, Christos Proukakis, Ester Kalef-Ezra

[DESCRIPTION]
There has been a growing recognition of the complexity of the human genome, and the role somatic variation plays in disease. The brain is particularly vulnerable to genomic mosaicism, likely arising during complex neurodevelopmental and ageing processes. However, current genomic technologies often lack the sensitivity to detect low-level genomic mosaics that could contribute to disease. An alternative cytogenetic method is DNA fluorescence in situ hybridisation (FISH), which allows for a targeted analysis of rare, disease-relevant copy number variants (CNVs). FISH can be subsequently combined with immunofluorescence to characterize somatic CNVs in specific cell populations based on specific protein marker expression. This protocol describes a method combining FISH with immunofluorescence, which we name immuno-FISH, for the detection of CNVs in the SNCA gene of patients with synucleinopathies, such as Parkinson’s disease (PD) and Multiple System Atrophy (MSA). This method is performed on nuclei isolated from frozen, human post-mortem brain tissue, which addresses potential sectioning artefacts and reduces protease digestion for epitope preservation. Our protocol is optimised to detect SOX10, a nuclear oligodendrocyte marker, and alpha-synuclein inclusions, which are frequently retained at the perinucleus in MSA (the so-called Papp-Lantos inclusions). This protocol also describes its use in affected PD and MSA brain regions such as the putamen, substantia nigra (SN) and cerebellum.

[GUIDELINES]
Intended purposes:

This protocol has been optimised for use on single-nuclei isolated from flash-frozen, human post-mortem brain tissue. It can be adapted to different SureFISH Agilent probes and antibodies for detecting nuclear markers.
  


[STEPS]
SECTION: Nuclei isolation from human post-mortem brain tissue using iodixanol gradient
1. Set the centrifuge to 4 °C. 
SECTION: Nuclei isolation from human post-mortem brain tissue using iodixanol gradient
2. Prepare ice-cold Carnoy’s fixative (3:1 Methanol: Glacial acetic acid) and 1X PBS.
SECTION: Nuclei isolation from human post-mortem brain tissue using iodixanol gradient
3. Isolate nuclei manually:
SECTION: Nuclei isolation from human post-mortem brain tissue using iodixanol gradient
3.1. See Table 1 for reagents and steps used for nuclei isolation. Refer to Kalef-Ezra, Perez-Rodriguez and Proukakis (dx.doi.org/10.17504/protocols.io.kxygxzjjov8j/v1) for details of the methods and solutions required for nuclei isolation implemented here. 
SECTION: Nuclei isolation from human post-mortem brain tissue using iodixanol gradient
3.2. Tissue guidelines: Use approximately 10-50 mg of brain tissue per nuclear suspension. Nuclei yield will vary between samples due to tissue collection, disease progression, and sub-regional differences between grey and white matter (cellular density and lipid composition among others).
 


SECTION: Nuclear yield check and visualization with DAPI (optional)
4. Resuspend the pellet containing the isolated nuclei in 500 µL of DAPI (1 1956 in 1x PBS working concentration).
SECTION: Nuclear yield check and visualization with DAPI (optional)
5. Leave the tube on a rotator disk for 20 min at 4 °C.

SECTION: Nuclear yield check and visualization with DAPI (optional)
6. Centrifuge at 800 x g, 5 min, 4 °C and remove the supernatant.

SECTION: Nuclear yield check and visualization with DAPI (optional)
7. Resuspend in 100-200 µL of 1X PBS.
SECTION: Nuclear yield check and visualization with DAPI (optional)
8. Use a haemocytometer and an epifluorescence microscope to estimate yield and visualise the spread of nuclei. The nuclear suspension should be evenly distributed, appear as single nuclei and free of large debris (see Figure 2 for examples).
  

SECTION: Nuclear yield check and visualization with DAPI (optional)
9. Centrifuge at 800 x g, 5 min to pellet nuclei and remove the supernatant.
SECTION: Nuclei fixation and preparation onto slide
10. Resuspend pellet containing the isolated nuclei in 1mL of pre-chilled Carnoy’s fixative and leave to fix on a rotator disk for 60 min at 4 °C.

SECTION: Nuclei fixation and preparation onto slide
11. Centrifuge 800 x g, 5 min and remove the supernatant. 
SECTION: Nuclei fixation and preparation onto slide
12. Resuspend pellet in 100-200 µL of Carnoy’s fixative by pipetting up and down. 
SECTION: Nuclei fixation and preparation onto slide
12.1. Optional: A   Flowmi cell strainer can be used to filter large clumps and debris.
SECTION: Nuclei fixation and preparation onto slide
13. Using a dropper or a pipette, place nuclear suspension onto an EprediaTM SuperFrost Plus Gold Adhesion slide and leave to evaporate for 20-60 min Room temperature.
 

SECTION: Nuclei fixation and preparation onto slide
14. Wash slides.
SECTION: Nuclei fixation and preparation onto slide
14.1. Wash slides for 5 min at Room temperature in an EasyDip slide staining jar containing 1X PBS. (1/2)
SECTION: Nuclei fixation and preparation onto slide
14.2. Wash slides for 5 min at Room temperature in an EasyDip slide staining jar containing 1X PBS. (2/2)
SECTION: Nuclei fixation and preparation onto slide
15. Check under microscope to assess the spread of nuclei before proceeding with immuno-FISH.
SECTION: FISH Pre-treatment
16. Prepare slide staining jars for FISH Pre-treatment according to Table 5 of Materials.
SECTION: FISH Pre-treatment
17. Place the water bath in a fume hood and set it to 72 °C.
SECTION: FISH Pre-treatment
18. Submerge the staining jar containing formamide solution into the water bath.
SECTION: FISH Pre-treatment
19. Set the oven to 37 °C and place the jar with dH2O inside (to which pepsin will be added afterwards), allow at least 30 min for solutions to reach the desired temperature.
SECTION: FISH Pre-treatment
20. Add HCl and pepsin to dH2O jar (according to Table 5 of Materials), then immediately place the slides in the pepsin solution for 5 min in the oven.
SECTION: FISH Pre-treatment
21. Transfer the slides to the PBS/MgCl2 solution and leave for 5 min at Room temperature.

SECTION: FISH Pre-treatment
22. Wash with 1X PBS once for 5 min at Room temperature.

SECTION: FISH Pre-treatment
23. Dehydrate the isolated nuclei in increasing concentrations of EtOH.
SECTION: FISH Pre-treatment
24. Allow the slides to air-dry for 10 min on the bench at Room temperature.
 

SECTION: FISH Pre-treatment
23.1. Dehydrate the isolated nuclei in 70% EtOH stored at Room temperature for 2 min.

SECTION: FISH Pre-treatment
23.2. Dehydrate the isolated nuclei in 90% EtOH stored at Room temperature for 2 min.

SECTION: FISH Pre-treatment
23.3. Dehydrate the isolated nuclei in 100% EtOH stored at Room temperature for 2 min.

SECTION: FISH Pre-treatment
25. Incubate the slides in the formamide solution for 3 min at 72 °C.
SECTION: FISH Pre-treatment
26. Dehydrate the nuclei in EtOH (pre-chilled at -20 °C).
SECTION: FISH Pre-treatment
26.1. Dehydrate the nuclei in 70% EtOH (pre-chilled at -20 °C) for 2 min at 
Room temperature.

SECTION: FISH Pre-treatment
26.2. Dehydrate the nuclei in 90% EtOH (pre-chilled at -20 °C) for 2 min at 
Room temperature.
SECTION: FISH Pre-treatment
26.3. Dehydrate the nuclei in 100% EtOH (pre-chilled at -20 °C) for 2 min at 
Room temperature.
SECTION: FISH Pre-treatment
27. Allow the slides to air-dry for 10 min on the bench at Room temperature.
 

SECTION: FISH Pre-treatment
28. Denature the FISH probe mixture for 5 min at 72 °C in the water bath.

SECTION: FISH Pre-treatment
29. Add 10 µL of the probe mixture to the slide, evenly distributing small droplets onto the nuclear suspension.

SECTION: FISH Pre-treatment
30. Place a 22mm x 22mm coverslip and seal the edges with rubber cement.
SECTION: FISH Pre-treatment
31. The FISH probes can be left to hybridise to DNA in a humidified box kept in the dark at 37 °C for 48-96 hrs.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
32. 
 
Place the water bath in a fume hood, then set temperature to 72 °C.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
33. Add Wash Buffer 1 at least 30 min in the water bath.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
34. Take out an aliquot of goat serum from -20 °C and leave to thaw at Room temperature.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
35. Peel off the rubber cement manually, soak the slides in 2X SSC for 10 min and then remove the coverslips from the slides.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
36. Wash the slides in FISH Wash Buffer 1 for 2 min at 72 °C in the water bath.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
37. Wash the slides in FISH Wash Buffer 2 for 1 min at Room temperature.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
38. Wash the slides.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
39. Hand-dry sections with tissue to remove PBS excess and create a hydrophobic barrier around the section using a Super PAP pen. 
 

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
40. Add 300 µL of the blocking solution and leave the slides in a humidified chamber for 60 min at Room temperature or 60 min at 4 °C.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
41. Remove the blocking solution excess and apply 150 µL of the primary antibody solution.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
42. Leave to incubate 2-4 hrs at Room temperature or 60 min at 4 °C.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
43. Wash the primary antibody solution off.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
44. Add 150 µL of the secondary antibody solution and leave to incubate for 60 min at Room temperature.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
45. Wash the secondary antibody off.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
46. Add 1 1956 DAPI (working concentration) to the slides for 20 min.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
47. Wash in 1X PBS for 5 min at Room temperature.

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
48. Add 200 µL of TrueBlack solution for 1 min.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
49. Quickly rinse the slides with 70% EtOH and then wash.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
50. Add 10-20 µL of Prolong Gold Anti-Fade solution and mount a 22mm x 22mm coverslip.
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
51. Leave the slides to dry in the dark 10 min at Room temperature before sealing the edges of the coverslip with nail varnish. Store them at 4 °C until use.
 
  
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
38.1. Wash the slides 10 min in 1X PBS at Room temperature. (1/3)

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
38.2. Wash the slides 10 min in 1X PBS at Room temperature. (2/3)

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
38.3. Wash the slides 10 min in 1X PBS at Room temperature. (3/3)

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
43.1. Wash the primary antibody solution off in 1X PBS for 10 min at Room temperature. (1/3)

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
43.2. Wash the primary antibody solution off in 1X PBS for 10 min at Room temperature. (2/3)
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
43.3. Wash the primary antibody solution off in 1X PBS for 10 min at Room temperature. (3/3)
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
45.1. Wash the secondary antibody off in 1X PBS for 10 min at Room temperature. (1/3)
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
45.2. Wash the secondary antibody off in 1X PBS for 10 min at Room temperature. (2/3)
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
45.3. Wash the secondary antibody off in 1X PBS for 10 min at Room temperature. (3/3)
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
49.1. Wash the slides in 1X PBS for 10 min at Room temperature. (1/3)

SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
49.2. Wash the slides in 1X PBS for 10 min at Room temperature. (2/3)
SECTION: FISH Post-hybridisation treatment &amp; immunofluorescence staining
49.3. Wash the slides in 1X PBS for 10 min at Room temperature. (3/3)