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Published on: December 11, 2021
A novel dual-release scaffold for fluorescent labels improves cyclic immunofluorescence
Thorge Reiber1,2, Christian Dose1, Dmytro A Yushchenko1
1Department of Chemical Biology, Miltenyi Biotec GmbH Friedrich-Ebert Straße 68 Bergisch Gladbach 51429 Germany dmytroy@miltenyi.com.
Insights
Researchers developed new fluorescent labels for cyclic immunofluorescence. These labels improve signal erasure, enabling more cycles for high-content imaging in cell biology and therapy development.
Area of Science:
- Cell Biology
- Biotechnology
- Immunology
Background:
- Cyclic immunofluorescence generates high-content imaging data for cell biology and therapeutics.
- Fluorescent labels are crucial for immunofluorescence quality and multiplexing capabilities.
- Current labels face limitations in signal erasure efficiency, restricting the number of staining cycles.
Purpose of the Study:
- To develop a novel fluorescent labelling strategy for enhanced cyclic immunofluorescence.
- To create labels with improved brightness and efficient signal erasure.
- To advance multiplexed cyclic immunofluorescence for deeper biological insights.
Main Methods:
- Antibodies were conjugated to a novel scaffold for enzymatic fluorophore cleavage.
- The scaffold comprised dextran decorated with single-stranded DNA (ssDNA).
- Dual enzymatic cleavage mechanisms (DNase and dextranase) were employed for signal erasure.
Main Results:
- The developed fluorescent labels demonstrated specific staining and high brightness.
- Labels performed effectively in flow cytometry and fluorescence microscopy.
- Dual enzymatic degradation significantly improved signal erasure from labelled epitopes.
Conclusions:
- The novel dual-release fluorescent labels enhance cyclic immunofluorescence.
- These labels offer high brightness and efficient, specific signal erasure.
- The strategy is expected to advance multiplexed cyclic immunofluorescence and cell biology research.
Abstract:
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.
