title: Labeling of active proteases in fresh-frozen tissues by topical application of quenched activity-based probes.
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.
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Nimali P
authors.initials: NP
authors.lastname: Withana
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Megan
authors.initials: M
authors.lastname: Garland
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Martijn
authors.identifier: http://orcid.org/0000-0001-8753-3528
authors.initials: M
authors.lastname: Verdoes
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Leslie O
authors.initials: LO
authors.lastname: Ofori
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Ehud
authors.initials: E
authors.lastname: Segal
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, USA.|Department of Chemical Systems and Biology, Stanford University School of Medicine, Stanford, California, USA.|Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
mesh_terms: D015925:Cryopreservation; D057075:Enzyme Assays; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D010447:Peptide Hydrolases / Q000378:metabolism*; D013194:Staining and Labeling / Q000191:economics / Q000379:methods*; D013696:Temperature; D016707:Tissue Fixation; D047368:Tumor Burden
chemical_list: D005456:Fluorescent Dyes; D010447:Peptide Hydrolases