浦肯野细胞
小脑
抗体
化学
神经科学
生物
免疫学
作者
Luke C. Bartelt,NULL AUTHOR_ID,NULL AUTHOR_ID,NULL AUTHOR_ID,NULL AUTHOR_ID,Paulina Jackowiak,Agnieszka Fiszer,NULL AUTHOR_ID,Albert R. La Spada,Paweł M. Świtoński
标识
DOI:10.1016/j.crmeth.2024.100816
摘要
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.
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