Tag-stabilized fluorescent protein binders for endogenous cell type-specific protein labelling

Protein labelling using genetically encoded fluorescent protein fusions has revolutionized the study of protein localization and function. However, visualizing endogenous proteins in specific cell types remains challenging, as existing approaches often require overexpression or laborious genetic engineering. To address these limitations, we developed a library of fluorescent protein binders using existing nanobodies and single-chain variable fragments that recognize fluorescent proteins and short peptide tags, allowing for modular, cell type-specific labelling of genetically tagged endogenous proteins. We found that fluorescent protein binders generate background fluorescence in the absence of the target protein, reducing signal specificity. To overcome this limitation, we generated destabilized fluorescent protein binders directed against GFP, ALFA, GCN4 peptide and V5. These binders display increased antigen-dependent stability and reduced background fluorescence, thereby improving the signal-to-noise ratio of endogenous protein visualization. We demonstrate the detection of endogenous proteins in drosophila larvae and embryos, zebrafish embryos, as well as in specific neuronal populations of the adult drosophila brain. In the brain, tissue-specific labelling revealed previously unknown protein expression and subcellular localization in sleep-regulatory neurons. Together, our panel of tag-stabilized fluorescent protein binders provides a versatile, ready-to-use toolkit for background-free, cell type-specific visualization of endogenous proteins.