Nanobody-fluorescent protein fusions for erasable immunostaining

Immunofluorescence (IF) staining is widely used to visualize biomolecules in cells and tissues. Conventional IF typically relies on fluorophore-conjugated antibodies, whose preparation requires chemical modification procedures that can compromise antibody performance and incur product loss during subsequent purification. In addition, iterative immunostaining depends on harsh stripping or fluorescence-quenching treatments that may damage sample architecture and epitope integrity. Here, we present protease-cleavable nanobody-fluorescent protein fusions (NFPFs) for reversible IF staining. Unlike fluorophore-labeled antibodies, NFPFs are genetically encoded and can be produced without post-synthetic chemical modification. By incorporating a tobacco etch virus (TEV) protease cleavage site between the nanobody and fluorescent protein, we engineered HER2- and EGFR-targeting NFPFs that retained high target specificity and enabled robust, concentration-dependent immunostaining of Capan-1 cells. TEV-mediated cleavage efficiently removed the fluorescent reporter while preserving cellular morphology and antigen accessibility, allowing subsequent staining of targets. Sequential labeling of distinct receptors demonstrated the feasibility of iterative immunostaining within the same specimen. Collectively, NFPFs provide a simple, scalable, and versatile approach for molecular imaging, enabling repeated rounds of immunostaining.