title: Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections.
abstract: Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.
authors.affiliation: Wolfson Institute for Biomedical Research, University College London.
authors.forename: Sarah
authors.initials: S
authors.lastname: Jolly
authors.affiliation: Wolfson Institute for Biomedical Research, University College London.
authors.forename: Alexander
authors.initials: A
authors.lastname: Fudge
authors.affiliation: Wolfson Institute for Biomedical Research, University College London.
authors.forename: Nigel
authors.initials: N
authors.lastname: Pringle
authors.affiliation: Wolfson Institute for Biomedical Research, University College London; w.richardson@ucl.ac.uk.
authors.forename: William D
authors.initials: WD
authors.lastname: Richardson
authors.affiliation: Wolfson Institute for Biomedical Research, University College London; huiliang.li@ucl.ac.uk.
authors.forename: Huiliang
authors.initials: H
authors.lastname: Li
mesh_terms: D000581:Amidohydrolases / Q000032:analysis*; D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D001921:Brain / Q000378:metabolism*; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D017628:Microglia / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D009836:Oligodendroglia / Q000378:metabolism
chemical_list: D000581:Amidohydrolases; C019751:aspartoacylase