Abstract
Purpose
The family of cathepsin proteases plays an important physiological role in both normal physiology and in the physiology of many human diseases. This activity, which is upregulated in many cancers, can be exploited for tumor imaging both in vivo and ex vivo. To characterize the behavior of a topically applied quenched fluorescent activity-based probe, GB119, ex vivo, we developed a basic immunohistochemistry technique to identify unquenched GB119 within tissue.
Procedures
Immunoblot assays were used to validate the utility of an anit-Cy5 antibody for the detection of unquenched GB119 generated by cathepsin-L. Following validation of the anti-Cy5 antibody, an immunohistochemical procedure was developed to detect the presence of unquenched GB119 in frozen sections of brain tumors derived from an orthotopic mouse model.
Results
These studies demonstrate that the anti-Cy5 antibody preferentially recognizes unquenched GB119 and that this differential can be used to identify the regions within the brain and the tumor that contained unquenched GB119. Using H&E staining and antibodies against other biochemical markers, it was further determined that unquenched GB119 was localized to the peri-tumor space and co-localized with cathepsin-L expression.
Conclusion
Our data indicate that this methodology allows high-resolution detection of unquenched GB119 that can be correlated with other immunohistological stains.
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Acknowledgments
Special thanks to Dr. Wilson for support of these studies by training and loan of his slide scanner and to Joe Meyers for his help in figure generation. This study was supported by a grant from the Coulter Foundation and the NFCR to J.P.B.
Conflict of Interest
JPB and MB are associated with Akrotome Imaging Inc. as both board members and co-founders. Akrotome is a company devoted to creating optical ABPs for imaging. Portions of the technology described in this report probe and have been licensed by Akrotome from both Case Western Reserve and Stanford Universities. This does not alter the authors' adherence to all the MIB policies on sharing data and materials.
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Walker, E., Gopalakrishnan, R., Bogyo, M. et al. Microscopic Detection of Quenched Activity-Based Optical Imaging Probes Using an Antibody Detection System: Localizing Protease Activity. Mol Imaging Biol 16, 608–618 (2014). https://doi.org/10.1007/s11307-014-0736-1
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DOI: https://doi.org/10.1007/s11307-014-0736-1