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Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes

Nature Protocols volume 11, pages 184–191 (2016)Cite this article

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Abstract

Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4–8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

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Figure 1: Structure of BMV109 and general protocol for tissue labeling using a quenched activity-based probe.
Figure 2: Topical staining of fresh-frozen human biopsy tissues with the optical cathepsin probe BMV109.
Figure 3: Inhibition of probe signal by the addition of the cathepsin inhibitor GB111-NH2 on fresh-frozen human biopsy tissues that have been labeled with the optical cathepsin probe BMV109.
Figure 4: Pictorial representation of protocol steps.

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Acknowledgements

We thank P. Chu from the Department of Pathology at Stanford University for assistance with processing the histology samples. We thank C.S. Kong and R. West from the Department of Pathology at Stanford University for intellectual input and pathological analysis of tissue samples. This work was funded by US National Institutes of Health grants R01 HL116307 and R01 EB005011 (to M.B.), by the Howard Hughes Medical Institute Medical Research Fellows program (to M.G.), and by the Stanford Medical Scientist Training Program (to M.G.).

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Author notes

  1. Martijn Verdoes

    Present address: Present address: Radboud University Medical Centre, Nijmegen, the Netherlands.,

  2. Nimali P Withana and Megan Garland: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, Stanford University School of Medicine, Stanford, California, USA

    Nimali P Withana, Megan Garland, Martijn Verdoes, Leslie O Ofori, Ehud Segal & Matthew Bogyo

  2. Department of Chemical Systems and Biology, Stanford University School of Medicine, Stanford, California, USA

    Matthew Bogyo

  3. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA

    Matthew Bogyo

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  1. Nimali P Withana
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Contributions

N.P.W. and M.G. designed and performed all experiments and drafted the manuscript. M.V. and L.O.O. designed and synthesized BMV109 and GB111-NH2 used in experiments. E.S. helped establish the tile scans and obtain tissue samples for analysis. M.B. developed and coordinated the project, analyzed the data and prepared and edited the manuscript.

Corresponding author

Correspondence to Matthew Bogyo.

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Withana, N., Garland, M., Verdoes, M. et al. Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes. Nat Protoc 11, 184–191 (2016). https://doi.org/10.1038/nprot.2016.004

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