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

IF 2.7 4区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Journal of Neuroscience Methods Pub Date : 2025-03-23 DOI:10.1016/j.jneumeth.2025.110434

Nicolette Tay , Ammar Alshammari , Simranpreet Kaur , Allison Pettit , Erica Mu , Anna Reid , Ingrid Winkler , Irina Vetter , Hana Starobova

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引用次数: 0

Abstract

Background

Dorsal root ganglia (DRGs) contain the cell bodies of sensory neurons and non-neuronal cells that play a role in the pathophysiology of painful inflammatory conditions, such as neuropathic pain. Immunohistochemistry (IHC) is a valuable tool for visualising and quantifying immune cell markers in DRGs, providing important insights into these mechanisms. However, isolating DRGs while preserving cell morphology for IHC staining is technically challenging due to their small size and location within the spinal column.

Objective

Using F4/80, a pan monocyte-macrophage marker, we present an optimised protocol for the fixation, harvesting, processing, and IHC staining of formalin-fixed-paraffin-embedded (FFPE) mouse DRGs. This method is designed to maintain tissue integrity and ensure compatibility with downstream histopathological analysis.

New Method

The entire spinal column of mouse was fixed in 10 % neutral-buffered formalin at room temperature for 24 h before DRG isolation. DRGs were processed for 9 h, and antigen retrieval was performed using proteinase K.

Results

The optimised immersion-fixation approach preserved cellular morphology and antigenicity, ensuring high-quality histological outcomes.

Comparison with Existing Methods

While transcardial perfusion remains the gold standard for tissue fixation, it is time-intensive, requires training and raises ethical concerns. Our optimised method of whole spinal column fixation with subsequent tissue isolation is non-invasive and reduces the time between death and fixation in comparison to post-isolation fixation. Additionally, it delivers histological quality likely comparable to that of perfusion-based techniques.

Conclusion

This protocol is supported by a grading system to help evaluate variables and select conditions best suited to their experimental goals.

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来源期刊

Journal of Neuroscience Methods 医学-神经科学

CiteScore

7.10

自引率

3.30%

发文量

226

审稿时长

52 days

期刊介绍： The Journal of Neuroscience Methods publishes papers that describe new methods that are specifically for neuroscience research conducted in invertebrates, vertebrates or in man. Major methodological improvements or important refinements of established neuroscience methods are also considered for publication. The Journal''s Scope includes all aspects of contemporary neuroscience research, including anatomical, behavioural, biochemical, cellular, computational, molecular, invasive and non-invasive imaging, optogenetic, and physiological research investigations.