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

Nat Protoc. 2016 Jan;11(1):184-91. doi: 10.1038/nprot.2016.004. Epub 2015 Dec 30.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cryopreservation*
  • Enzyme Assays
  • Fluorescent Dyes / metabolism*
  • Humans
  • Immunohistochemistry
  • Microscopy, Fluorescence
  • Peptide Hydrolases / metabolism*
  • Staining and Labeling / economics
  • Staining and Labeling / methods*
  • Temperature
  • Tissue Fixation
  • Tumor Burden


  • Fluorescent Dyes
  • Peptide Hydrolases