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Summary: Immunofluorescent labeling is a widely used method to visualize endogenous proteins. It can be expensive and difficult to stain mouse embryonic stem cells (mESCs) because they require expensive growth media, prefer specific substrates, grow in 3D, and have loose cell-substrate adhesion. Here we propose a half-a-day, cheap, easy-to-follow, and reproducible protocol for immunofluorescence of mESCs. This protocol has been streamlined to allow a fast visualization of the investigated proteins, and we provide tips specific to stem cell culture.For complete details on the use and execution of this protocol, please refer to Chaigne et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.