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Protocol to image and quantify nuclear pore complexes using high-resolution laser scanning confocal microscopy.

Jocelyn D Mich-Basso1, Bernhard Kühn1

  • 1Division of Cardiology, Pediatric Institute for Heart Regeneration and Therapeutics (I-HRT), UPMC Children's Hospital of Pittsburgh, 4401 Penn Avenue, Pittsburgh, PA 15224, USA; Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA.

STAR Protocols
|August 31, 2023
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Summary

This study provides a protocol to image and quantify nuclear pore complexes (NPCs), crucial for cell communication and chromatin organization. The method uses immunofluorescence and confocal microscopy for accurate NPC counting in cells.

Keywords:
Cell BiologyMicroscopy

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Microscopy

Background:

  • Nuclear pore complexes (NPCs) are essential channels for nucleocytoplasmic transport.
  • NPCs play a critical role in regulating gene expression and chromatin organization.
  • Accurate quantification of NPCs is vital for understanding cellular processes.

Purpose of the Study:

  • To present a detailed protocol for imaging and quantifying nuclear pore complexes in cells.
  • To provide a reproducible method for researchers studying NPC dynamics and function.

Main Methods:

  • Cell culture and plating.
  • Immunofluorescence staining for NPC proteins.
  • Confocal microscopy for 3D visualization.
  • Digital thresholding with human supervision for quantification.

Main Results:

  • The protocol enables precise imaging of NPCs within cellular structures.
  • Quantification of NPC numbers is achieved through a supervised digital analysis approach.
  • The method allows for detailed 3D data analysis of NPC distribution.

Conclusions:

  • This protocol offers a reliable method for quantifying nuclear pore complexes.
  • The presented technique facilitates research into NPC roles in cellular communication and chromatin structure.