Microscopic detection of quenched activity-based optical imaging probes using an antibody detection system: localizing protease activity.
OBJECTIVE
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.


METHODS
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.


CONCLUSIONS
Our data indicate that this methodology allows high-resolution detection of unquenched GB119 that can be correlated with other immunohistological stains.
Department of Radiology, Case Center for Imaging Research, Case Western Reserve University, 11100 Euclid Ave, Cleveland, OH, 44106-5056, USA.
Ethan
E
Walker
Ramamurthy
R
Gopalakrishnan
Matthew
M
Bogyo
James P
JP
Basilion
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D001932:Brain Neoplasms / Q000378:metabolism; D002232:Carbocyanines; D056668:Cathepsin L / Q000378:metabolism*; D002457:Cell Extracts; D005260:Female; D005456:Fluorescent Dyes; D008819:Mice, Nude; D008853:Microscopy / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D013194:Staining and Labeling
D000906:Antibodies; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D002457:Cell Extracts; D005456:Fluorescent Dyes; D056668:Cathepsin L