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Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay
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JoVE Journal Medicina
Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

DOI:
10.3791/59398-v

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08:40 min

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May 02, 2019

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,
1Department of Internal Medicine, Division of Metabolism, Endocrinology and Diabetes,University of Michigan, Ann Arbor, 2VA Ann Arbor Healthcare System

Capitoli

  • 00:04Titolo
  • 00:32Human Islet Sample Single Cell Dissociation
  • 02:14Single Cell Adherence and Fixation and Immunohistochemistry
  • 03:24Proximity Ligation Assay (PLA)
  • 04:38Imaging
  • 06:00PLA Interaction Quantification
  • 07:18Results: Representative Beta Cell Mitophagy Analysis
  • 08:08Conclusion

Summary

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This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Tags

MitophagyProximity Ligation AssayHuman Pancreatic Beta CellsEndogenous Mitophagy ComplexesImmunohistochemistryUbiquitin-specific Peptidase 8Neuregulin Receptor Degradation Protein-1Beta Cell Marker

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