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3D Immunofluorescent Image Colocalization Quantification in Mouse Epiblast Stem Cells.

Joshua G Dierolf1, Andrew J Watson1,2,3, Dean H Betts4,5,6

  • 1Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, ON, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|April 29, 2022
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Summary

This study optimizes 3D colony architecture and protein localization in mouse epithelial stem cells (mESCs) using confocal microscopy. These methods efficiently assess nuclear and cytoplasmic protein distribution in various stem cell types.

Keywords:
ColocalizationConfocal microscopyCytoplasmicImmunofluorescenceNuclearPrimed pluripotencymEpiSC

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Area of Science:

  • Cell Biology
  • Microscopy Techniques
  • Stem Cell Research

Background:

  • Understanding protein localization is crucial for stem cell differentiation.
  • Current methods for assessing protein localization can be complex and time-consuming.

Purpose of the Study:

  • To detail optimized protocols for 3D morphological topography and nuclear protein localization analysis.
  • To demonstrate efficient colocalization assessment in mouse epithelial stem cells (mESCs), mouse epiblast-like cells (mEpiLCs), and mouse epithelial-like stem cells (mEpiSCs).

Main Methods:

  • Co-immunofluorescent confocal microscopy for 3D morphological topography and protein localization.
  • Orthogonal colocalization assessment to evaluate nuclear and cytoplasmic protein distribution.
  • Application of optimized protocols to mESCs, mEpiLCs, and mEpiSCs.

Main Results:

  • Successful optimization of 3D colony architecture analysis.
  • Efficient demonstration of nuclear and cytoplasmic protein localization in mEpiSCs.
  • High-throughput colocalization assessment achieved using the developed protocols.

Conclusions:

  • The presented optimized protocols enable efficient and accurate 3D morphological and protein localization analysis in stem cells.
  • These methods facilitate a deeper understanding of protein distribution and its role in stem cell function.
  • The protocols are applicable across different mouse stem cell subtypes.