Assembly and operation of a cooling stage to immobilizeC. eleganson their culture plates

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans ( C. elegans ) but requires strong animal immobilization. Unfortunately, most current immobilization techniques require substantial manual effort, rendering high-resolution imaging low-throughput. We greatly simplify the C. elegans immobilization procedure by using a cooling approach that can easily immobilize entire populations of C. elegans directly on their cultivation plates. The cooling stage can establish and maintain a wide range of temperatures on the cultivation plate and ensures even temperature distribution across the agarose surface. In this article, we document the whole process of building the cooling stage from scratch. We envision that a typical researcher can build an operational cooling stage in their lab following our protocol without difficulty. We show how to utilize the cooling stage under three protocols, which have advantages for different experiments. We also show an example cooling profile of the stage as it approaches its final temperature and include some helpful tips in using cooling immobilization.