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A comprehensive protocol for simplified mouse DRG fixation, processing and F4/80 immunohistochemistry: Overcoming

Nicolette Tay1, Ammar Alshammari2, Simranpreet Kaur3

  • 1School of Pharmacy and Pharmaceutical Sciences, The University of Queensland, Woolloongabba, QLD, Australia.

Journal of Neuroscience Methods
|March 25, 2025
PubMed
Summary

We optimized a method for fixing and processing mouse dorsal root ganglia (DRGs) for immunohistochemistry (IHC). This technique preserves tissue integrity and cell morphology, enabling better analysis of neuropathic pain mechanisms.

Keywords:
F4/80Immersion fixationImmunohistochemistryOptimised DRG morphologyOptimised tissue harvest protocolWhole spinal column DRG isolation

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Area of Science:

  • Neuroscience
  • Immunology
  • Histopathology

Background:

  • Dorsal root ganglia (DRGs) are crucial in neuropathic pain.
  • Immunohistochemistry (IHC) visualizes immune cells in DRGs.
  • DRG isolation for IHC is challenging due to size and location.

Purpose of the Study:

  • Optimize fixation, harvesting, processing, and IHC staining of mouse DRGs.
  • Utilize F4/80, a monocyte-macrophage marker, for analysis.
  • Ensure tissue integrity and compatibility with histopathology.

Main Methods:

  • Whole mouse spinal column fixation in 10% neutral-buffered formalin.
  • DRG isolation after 24h fixation.
  • 9h processing and proteinase K antigen retrieval.

Main Results:

  • Optimized immersion-fixation preserved cellular morphology and antigenicity.
  • Achieved high-quality histological outcomes.
  • Method is non-invasive and reduces time to fixation.

Conclusions:

  • The protocol provides high-quality histological outcomes.
  • It is comparable to perfusion-based techniques.
  • A grading system aids in selecting optimal experimental conditions.