Neuronal tracing with DiI: decalcification, cryosectioning, and photoconversion for light and electron microscopic...

Area of Science:

• Neuroscience
• Cell Biology
• Histology

Background:

• 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is a lipophilic fluorescent dye used for tracing neuronal pathways.
• DiI diffusion and fluorescence properties are retained in aldehyde-fixed tissues, enabling post-mortem neuronal tracing.
• Existing DiI tracing methods have limitations in decalcification, sectioning, and ultrastructural analysis.

Purpose of the Study:

• To describe modifications of the DiI tracing method for enhanced neuronal analysis.
• To enable DiI tracing in decalcified and sectioned neural tissues.
• To adapt DiI labeling for electron microscopy through photoconversion.

Main Methods:

• Tissue decalcification using EDTA at 37°C while DiI diffuses.
• Sectioning of DiI-injected tissue on a cryostat with minimal dye spread.
• Photoconversion of DiI label in cryosections and Vibratome sections to a diaminobenzidine (DAB) reaction product.

Main Results:

• DiI tracing is feasible in decalcified tissues, expanding applications to bony-encased neural structures.
• A protocol for cryosectioning DiI-labeled tissue minimizes dye diffusion in dried sections.
• Photoconverted DiI label yields an electron-dense DAB product, allowing ultrastructural analysis of neurons and their processes.
• DiI localizes to internal organelles and microsomal structures in fixed tissue.
• Photoconversion is compatible with gold immunocytochemistry.

Conclusions:

• Modified DiI tracing methods significantly enhance its utility for post-mortem neuronal analysis.
• The technique allows for detailed ultrastructural investigation of labeled neurons, including intracellular localization.
• These advancements provide robust methods for long-term preservation and high-resolution imaging of neural circuits.