An optimized procedure of EZ clearing protocol for high reproducibility clearing and labeling

We present an optimized tissue clearing procedure that preserves fluorescence markers and enables immunolabeling in tissue such as mouse sciatic nerve, gut and spinal cord. By refining the original protocol, we ensure stable clearing across various tissues. Key modifications include maintaining tetrahydrofuran (THF) solvent under inert atmosphere, adjusting delipidation based on tissue type, and enhancing antibody penetration via an extracellular matrix loosening strategy. Applying iDISCO+ immunolabeling protocol, we achieve uniform staining in thin tissues. Finally, we confirm long-term endogenous fluorescence retention, ensuring stability for extended imaging and analysis.