Targeted detection of SNCA CNVs in SOX10+ nuclei from oligodendrocytes containing alpha-synuclein inclusions isolated from human post-mortem brain
Ester Kalef-Ezra, Christos Proukakis, Caoimhe Morley, Diego-Perez Rodriguez, Monica Emili Garcia-Segura
Disclaimer
This protocol was adapted from the following:
Garcia-Segura, M.E., Perez-Rodriguez, D. and Proukakis, C. (2022) ‘Combined fluorescence in situ hybridization (FISH) and immunofluorescence for the targeted detection of somatic copy number variants in Synucleinopathies’, Neuromethods, pp. 229–243.
Ester Kalef-Ezra, Diego Perez-Rodriguez, Christos Proukakis. Manual isolation of nuclei from human brain using CellRaft device and single nucleus Whole Genome Amplification. Protocols.io ([https://protocols.io/view/manual-isolation-of-nuclei-from-human-brain-using-cx4mxqu6).](https://protocols.io/view/manual-isolation-of-nuclei-from-human-brain-usingcx4mxqu6).)
Abstract
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.
Attachments
Steps
Nuclei isolation from human post-mortem brain tissue using iodixanol gradient
Set the centrifuge to 4°C.
Prepare ice-cold Carnoy’s fixative (3:1 Methanol: Glacial acetic acid) and 1X PBS.
Isolate nuclei manually:
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.
Tissue guidelines: Use approximately 10-50mg 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).
Nuclear yield check and visualization with DAPI (optional)
Resuspend the pellet containing the isolated nuclei in 500µL of DAPI (1 in 1x PBS working concentration).
Leave the tube on a rotator disk for 0h 20m 0s at 4°C.
Centrifuge at 800x g,4°C and remove the supernatant.
Resuspend in 100-200µL of 1X PBS.
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).
Centrifuge at 800x g to pellet nuclei and remove the supernatant.
Nuclei fixation and preparation onto slide
Resuspend pellet containing the isolated nuclei in 1mL of pre-chilled Carnoy’s fixative and leave to fix on a rotator disk for 1h 0m 0s at 4°C.
Centrifuge 800x g and remove the supernatant.
Resuspend pellet in 100-200µL of Carnoy’s fixative by pipetting up and down.
Optional: A 70 µm Flowmi cell strainer can be used to filter large clumps and debris.
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.
Wash slides.
Wash slides for 0h 5m 0s at Room temperature in an EasyDip slide staining jar containing 1X PBS. (1/2)
Wash slides for 0h 5m 0s at Room temperature in an EasyDip slide staining jar containing 1X PBS. (2/2)
Check under microscope to assess the spread of nuclei before proceeding with immuno-FISH.
FISH Pre-treatment
Prepare slide staining jars for FISH Pre-treatment according to Table 5 of Materials.
Place the water bath in a fume hood and set it to 72°C.
Submerge the staining jar containing formamide solution into the water bath.
Set the oven to 37°C and place the jar with dH2O inside (to which pepsin will be added afterwards), allow at least 0h 30m 0s for solutions to reach the desired temperature.
Add HCl and pepsin to dH2O jar (according to Table 5 of Materials), then immediately place the slides in the pepsin solution for 0h 5m 0s in the oven.
Transfer the slides to the PBS/MgCl2 solution and leave for 0h 5m 0s at Room temperature.
Wash with 1X PBS once for 0h 5m 0s at Room temperature.
Dehydrate the isolated nuclei in increasing concentrations of EtOH.
Dehydrate the isolated nuclei in 70% EtOH stored at Room temperature for 0h 2m 0s.
Dehydrate the isolated nuclei in 90% EtOH stored at Room temperature for 0h 2m 0s.
Dehydrate the isolated nuclei in 100% EtOH stored at Room temperature for 0h 2m 0s.
Allow the slides to air-dry for 0h 10m 0s on the bench at Room temperature.
Incubate the slides in the formamide solution for 0h 3m 0s at 72°C.
Dehydrate the nuclei in EtOH (pre-chilled at -20°C).
Dehydrate the nuclei in 70% EtOH (pre-chilled at -20°C) for 0h 2m 0s at
Room temperature .
Dehydrate the nuclei in 90% EtOH (pre-chilled at -20°C) for 0h 2m 0s at
Room temperature .
Dehydrate the nuclei in 100% EtOH (pre-chilled at -20°C) for 0h 2m 0s at
Room temperature .
Allow the slides to air-dry for 0h 10m 0s on the bench at Room temperature.
Denature the FISH probe mixture for 0h 5m 0s at 72°C in the water bath.
Add 10µL of the probe mixture to the slide, evenly distributing small droplets onto the nuclear suspension.
Place a 22mm x 22mm coverslip and seal the edges with rubber cement.
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.
FISH Post-hybridisation treatment & immunofluorescence staining
72°C. Add Wash Buffer 1 at least 0h 30m 0s in the water bath.
Take out an aliquot of goat serum from -20°C and leave to thaw at Room temperature.
Peel off the rubber cement manually, soak the slides in 2X SSC for 0h 10m 0s and then remove the coverslips from the slides.
Wash the slides in FISH Wash Buffer 1 for 0h 2m 0s at 72°C in the water bath.
Wash the slides in FISH Wash Buffer 2 for 0h 1m 0s at Room temperature.
Wash the slides.
Wash the slides 0h 10m 0s in 1X PBS at Room temperature. (1/3)
Wash the slides 0h 10m 0s in 1X PBS at Room temperature. (2/3)
Wash the slides 0h 10m 0s in 1X PBS at Room temperature. (3/3)
Hand-dry sections with tissue to remove PBS excess and create a hydrophobic barrier around the section using a Super PAP pen.
Add 300µL of the blocking solution and leave the slides in a humidified chamber for 1h 0m 0s at Room temperature or 1h 0m 0s at 4°C.
Remove the blocking solution excess and apply 150µL of the primary antibody solution.
Leave to incubate 2-4 hrs at Room temperature or 1h 0m 0s at 4°C.
Wash the primary antibody solution off.
Wash the primary antibody solution off in 1X PBS for 0h 10m 0s at Room temperature. (1/3)
Wash the primary antibody solution off in 1X PBS for 0h 10m 0s at Room temperature. (2/3)
Wash the primary antibody solution off in 1X PBS for 0h 10m 0s at Room temperature. (3/3)
Add 150µL of the secondary antibody solution and leave to incubate for 1h 0m 0s at Room temperature.
Wash the secondary antibody off.
Wash the secondary antibody off in 1X PBS for 0h 10m 0s at Room temperature. (1/3)
Wash the secondary antibody off in 1X PBS for 0h 10m 0s at Room temperature. (2/3)
Wash the secondary antibody off in 1X PBS for 0h 10m 0s at Room temperature. (3/3)
Add 1 DAPI (working concentration) to the slides for 0h 20m 0s.
Wash in 1X PBS for 0h 5m 0s at Room temperature.
Add 200µL of TrueBlack solution for 0h 1m 0s.
Quickly rinse the slides with 70% EtOH and then wash.
Wash the slides in 1X PBS for 0h 10m 0s at Room temperature. (1/3)
Wash the slides in 1X PBS for 0h 10m 0s at Room temperature. (2/3)
Wash the slides in 1X PBS for 0h 10m 0s at Room temperature. (3/3)
Add 10-20µL of Prolong Gold Anti-Fade solution and mount a 22mm x 22mm coverslip.
Leave the slides to dry in the dark 0h 10m 0s at Room temperature before sealing the edges of the coverslip with nail varnish. Store them at 4°C until use.
