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Targeted detection of SNCA CNVs in SOX10+ nuclei from oligodendrocytes containing alpha-synuclein inclusions isolated from human post-mortem brain
Output Details
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.
Identifier (DOI)
10.17504/protocols.io.5jyl8p6r7g2w/v1