Protocol for the three-dimensional analysis of rodent skeletal muscle

Area of Science:

• Biomedical Engineering
• Cell Biology
• Anatomy

Background:

• Confocal imaging offers advanced cellular spatial data analysis within tissues.
• Characterizing cell types in skeletal muscle requires robust sample preparation techniques.

Purpose of the Study:

• To present a protocol for preparing optically cleared extensor digitorum longus (EDL) skeletal muscle samples.
• To enable detailed confocal imaging and computational analysis of cellular structures within muscle tissue.

Main Methods:

• Protocol involves perfusion fixation and tissue clearing of skeletal muscle samples.
• Includes detailed steps for image acquisition, computational analysis, sample segmentation, and 3D rendering.
• Applicable to rodent skeletal muscle, specifically the EDL.

Main Results:

• Successfully prepared optically cleared EDL skeletal muscle samples.
• Demonstrated suitability for high-resolution confocal imaging and subsequent computational analysis.
• Enabled detailed visualization and characterization of muscle satellite cells and capillary endothelial cells.

Conclusions:

• The developed protocol is effective for preparing skeletal muscle for advanced imaging.
• Facilitates the study of cellular spatial organization and cell types within muscle tissue.
• Supports research in muscle stem cells and vascular biology.