Protocol for automated analysis of oil red O-stained Caenorhabditis elegans using Redwormpix, a Python-based application.

Oil red O (ORO) staining is a widely used histological technique for visualizing neutral lipids in Caenorhabditis elegans. However, quantitative image analysis of ORO-stained worms remains largely manual, time-consuming, and prone to human error. Here, we present a protocol for automating the quantification of ORO staining using Redwormpix, an open-source Python-based application. We describe steps for installing software, transferring images, and running the application. We then detail procedures for manual validation and exporting results.