Iodine Staining of Glycogen Storage in Caenorhabditis elegans

Glycogen is a highly conserved macromolecule across species, and its visualization provides critical insights into both physiological processes and disease states. Existing approaches for glycogen imaging in Caenorhabditis elegans rely primarily on traditional microscopy slides, which introduce variability in image acquisition and downstream data analysis, limit throughput, and require substantial hands-on time and technical expertise. Here, we present a standardized, cost-effective, and high-throughput imaging method that enables efficient visualization and quantification of glycogen in C. elegans. Our approach utilizes a custom-designed three-dimensional pad containing two to four chambers, allowing control and experimental samples to be processed simultaneously under identical conditions. Worms are exposed to iodine crystals, ensuring uniform staining while minimizing reagent use and handling variability. Imaging is performed using a simple binocular microscope, and analysis is conducted in Fiji, making the workflow accessible to laboratories with minimal specialized equipment or training. This method also reduces technical variability, shortens turnaround time, and requires only basic reagents and expertise, making it well-suited for both research and teaching laboratories. Importantly, the platform is readily adaptable to other nematode species and scalable for large-scale genetic or pharmacological screening applications. Together, this workflow minimizes technical variability and provides a robust platform for comparative glycogen analysis in C. elegans. Key features • Standardized glycogen staining method allows less data variability. • Provides a quantitative tool to measure glycogen buildup in C. elegans. • Requires a customized pad for imaging. • Macro is available for batch processing.