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Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy

Nature Protocols volume 17, pages 819–846 (2022)Cite this article

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Abstract

Fluorescence microscopy is a vital tool in biomedical research but faces considerable challenges in achieving uniform or bright labeling. For instance, fluorescent proteins are limited to model organisms, and antibody conjugates can be inconsistent and difficult to use with thick specimens. To partly address these challenges, we developed a labeling protocol that can rapidly visualize many well-contrasted key features and landmarks on biological specimens in both thin and thick tissues or cultured cells. This approach uses established reactive fluorophores to label a variety of biological specimens for cleared-tissue microscopy or expansion super-resolution microscopy and is termed FLARE (fluorescent labeling of abundant reactive entities). These fluorophores target chemical groups and reveal their distribution on the specimens; amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible amines on proteins, while hydrazide fluorophores target oxidized carbohydrates. The resulting stains provide signals analogous to traditional general histology stains such as H&E or periodic acid–Schiff but use fluorescent probes that are compatible with volumetric imaging. In general, the stains for FLARE are performed in the order of carbohydrates, amine and DNA, and the incubation time for the stains varies from 1 h to 1 d depending on the combination of stains and the type and thickness of the biological specimens. FLARE is powerful, robust and easy to implement in laboratories that already routinely do fluorescence microscopy.

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Fig. 1: Overview of FLARE for fluorescent labeling of biological samples.
Fig. 2: A summary of suggested workflows when combining FLARE with DNA FISH and immunofluorescence for clearing or expansion.
Fig. 3: Comparison of fixation conditions for expanded RPE cells stained by FLARE.
Fig. 4: FLARE staining of 5-µm-thick FFPE human tissue microarrays.
Fig. 5: Compatibility of FLARE with diverse procedures.
Fig. 6: Gelation chamber setup.
Fig. 7: A step-by-step illustration of the FLARE procedure for the expanded sample.
Fig. 8: FLARE labeling of ~1-mm-thick kidney tissue.
Fig. 9: A step-by-step illustration of the FLARE procedure for the optically cleared sample.
Fig. 10: Demonstration of expansion uniformity using a MAP-based protocol.
Fig. 11: Proper sample orientation during staining and image acquisition.
Fig. 12: Demonstration of the inner filter effect for densely labeled samples.

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Data availability

Data other than those presented in the paper and in the authors’ previous paper3 are available from the corresponding author upon request.

Code availability

The custom Wolfram Mathematica scripts used for stitching tiling images (Fig. 4a,h) are available as Supplementary Code.

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Acknowledgements

This work was supported by the University of Washington, NIDDK Diabetic Complications Consortium grants DK076169 and DK115255 (J.C.V.), NIH grants R01 MH115767 (J.C.V.), R01 CA244170 (J.T.C.L.), R01 EB031002 (J.T.C.L.) and K99 CA240681 (A.K.G.), and DoD PCRP grant W81XWH-18-10358 (J.T.C.L.). NW BioTrust, a core service for patient consenting, and NWBioSpecimen, a core service for procurement and annotation of research biospecimens, are supported by National Cancer Institute grant P30 CA015704 (G. Gilliland, principal investigator (PI)), Institute of Translational Health Sciences grant UL1 TR000423 (M. Disis, PI), the University of Washington School of Medicine and Department of Pathology, and Fred Hutchinson Cancer Research Center.

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Author notes

  1. These authors contributed equally: Min Yen Lee, Chenyi Mao.

Authors and Affiliations

  1. Department of Chemistry, University of Washington, Seattle, WA, USA

    Min Yen Lee, Chenyi Mao, Marcus A. Woodworth, Aaron R. Halpern, Adilijiang Ali & Joshua C. Vaughan

  2. Department of Mechanical Engineering, University of Washington, Seattle, WA, USA

    Adam K. Glaser & Jonathan T. C. Liu

  3. Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA

    Jonathan T. C. Liu

  4. Department of Bioengineering, University of Washington, Seattle, WA, USA

    Jonathan T. C. Liu

  5. Department of Physiology and Biophysics, University of Washington, Seattle, WA, USA

    Joshua C. Vaughan

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Contributions

C.M., M.Y.L., A.K.G., M.A.W., J.T.C.L. and J.C.V. designed the methodology. C.M., M.Y.L., A.K.G., M.A.W. and A.R.H performed the experiments and analysis. C.M., M.Y.L., A.K.G., M.A.W., J.T.C.L. and J.C.V. wrote the paper, and all authors commented on the manuscript. J.C.V. supervised the project.

Corresponding author

Correspondence to Joshua C. Vaughan.

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Competing interests

J.T.C.L and A.K.G are co-founders and hold equity in Lightspeed Microscopy Inc., of which J.T.C.L is also a board member. The other authors declare no competing interests.

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Key references using this protocol

Mao, C. et al. Sci. Adv. 6, eaba4542 (2020): https://doi.org/10.1126/sciadv.aba4542

Chen, Y. et al. Biomed. Opt. Express 10, 1257 (2019): https://doi.org/10.1364/BOE.10.001257

Supplementary information

Supplementary information

Supplementary Method 1, Supplementary Figs. 1–4, Supplementary Tables 1 and 2 and the first frame of Supplementary Video 1.

Reporting Summary

Supplementary Video 1

Open-top light-sheet imaging of a ~1-mm-thick mouse kidney section. (Adapted with permission from ref. 3, AAAS, under a Creative Commons license CC BY-NC 4.0.)

Supplementary Code 1

Custom Wolfram Mathematica scripts used for stitching tiling images.

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Lee, M.Y., Mao, C., Glaser, A.K. et al. Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy. Nat Protoc 17, 819–846 (2022). https://doi.org/10.1038/s41596-021-00667-2

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