Protocol for reproducible EZ clearing and labeling, including optimized steps and quantified fluorescence retention on mouse tissue.

Here, we present a protocol for preserving fluorescent markers using tissue clearing and immunolabeling in mouse tissue such as sciatic nerve, gut, and spinal cord. We describe steps for maintaining tetrahydrofuran (THF) under an inert atmosphere, EZ clearing, immunolabeling, imaging with custom lightsheet microscopy setups, and post-processing. This protocol was refined to ensure stable and reliable clearing across various tissue types. Key improvements include inert atmosphere THF handling, tissue-specific delipidation, and enhanced antibody penetration via extracellular matrix loosening.