Spinal Cord Research Help

Fluorescent whole-mount method for visualizing 3-dimensional relationships in intact and regenerating adult newt spinal cords

Dev Dyn, 2010 · DOI: 10.1002/dvdy.22441 · Published: November 1, 2010

Regenerative MedicineNeurologyResearch Methodology & Design

Simple Explanation

Adult newts can regenerate their spinal cords after injury. This study developed a method to visualize the cellular and molecular events during this regeneration in 3D. The method uses fluorescent probes and confocal microscopy on whole-mount preparations of newt spinal cords. The technique was optimized by adjusting fixation, tissue trimming, probe penetration, and clearing methods.

Study Duration
Not specified
Participants
Adult newts, Notophthalmus viridescens
Evidence Level
Not specified

Key Findings

  • 1
    PLP with 0.5% PFA fixative provided the best streptavidin probe penetration while preserving cellular morphology.
  • 2
    Increasing the amount of Triton X-100 detergent to 2%, longer incubation times with primary and secondary antibodies, and improved mixing of antibody solution enhanced antibody labeling.
  • 3
    Formamide bleach was more effective than modified Dent's bleach for clearing pigment from whole-mount specimens.

Research Summary

This paper presents a method for visualizing 3D relationships in intact and regenerating adult newt spinal cords using fluorescent probes and confocal microscopy. The method involves fixation, decalcification, bleaching, embedding, sectioning, permeabilization, fluorescent labeling, and clearing. The authors optimized parameters for each step, including fixatives, detergents, incubation times, and clearing agents, to achieve optimal probe penetration and image quality.

Practical Implications

Research Tool

The method provides a valuable tool for studying spinal cord regeneration in newts and other adult tissues.

Adaptability

The techniques are adaptable to study 3D relationships in other adult tissues.

Clarity Improvement

The optimized clearing and labeling techniques improve the clarity and detail of 3D tissue imaging.

Study Limitations

  • 1
    The heterogeneous nature of the spinal column made it difficult to obtain sections thinner than 100 μm.
  • 2
    BABB, although effective for clearing, is a caustic solution and makes the tissue brittle.
  • 3
    SDS disrupted the morphology of the spinal cord without improving antibody penetration.

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