A novel dual-release scaffold for fluorescent labels improves cyclic immunofluorescence†

Cyclic immunofluorescence is a powerful method to generate high-content imaging datasets for investigating cell biology and developing therapies. This method relies on fluorescent labels that determine the quality of immunofluorescence and the maximum number of staining cycles that can be performed. Here we present a novel fluorescent labelling strategy, based on antibodies conjugated to a scaffold containing two distinct sites for enzymatic cleavage of fluorophores. The scaffold is composed of a dextran decorated with short ssDNA that upon hybridization with complementary dye-modified oligos result in fluorescent molecules. The developed fluorescent labels exhibit specific staining and remarkable brightness in flow cytometry and fluorescence microscopy. We showed that the combination of DNase-mediated degradation of DNA and dextranse-mediated degradation of the dextran as two complementary enzymatic release mechanisms in one molecule, improves signal erasure from labelled epitopes. We envision that such dual-release labels with high brightness and efficient and specific erasure will advance multiplexed cyclic immunofluorescence approaches and thereby will contribute to gaining new insights in cell biology.