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Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue
Published on: July 10, 2012
Antibody-assisted selective isolation of Purkinje cell nuclei from mouse cerebellar tissue
Luke C Bartelt1, Mouad Fakhri2, Grazyna Adamek2
1University Program in Genetics & Genomics, Duke University Medical Center, Durham, NC 27710, USA; Departments of Pathology & Laboratory Medicine, Neurology, Biological Chemistry, and Neurobiology & Behavior, University of California, Irvine, Irvine, CA 92697, USA; Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Abstract:
We developed a method that utilizes fluorescent labeling of nuclear envelopes alongside cytometry sorting for the selective isolation of Purkinje cell (PC) nuclei. Beginning with SUN1 reporter mice, we GFP-tagged envelopes to confirm that PC nuclei could be accurately separated from other cell types. We then developed an antibody-based protocol to make PC nuclear isolation more robust and adaptable to cerebellar tissues of any genotypic background. Immunofluorescent labeling of the nuclear membrane protein RanBP2 enabled the isolation of PC nuclei from C57BL/6 cerebellum. By analyzing the expression of PC markers, nuclear size, and nucleoli number, we confirmed that our method delivers a pure fraction of PC nuclei. To demonstrate its applicability, we isolated PC nuclei from spinocerebellar ataxia type 7 (SCA7) mice and identified transcriptional changes in known and new disease-associated genes. Access to pure PC nuclei offers insights into PC biology and pathology, including the nature of selective neuronal vulnerability.
