Fig. 2

Competitive tissue-ABPP demonstrating that TAMRA-FP reports SH activity in glioma brain sections. Sections were pretreated for 1 h with the indicated concentrations of various serine-nucleophile targeting inhibitors and processed for TAMRA-FP labeling and confocal fluorescence imaging as detailed in Materials and Methods. Sections from two individual rats bearing the tumor were used and images from two independent experiments are shown as separate panels with DMSO control included in both experiments. Desthiobiotin-FP (5 μM) efficiently inhibits TAMRA-FP labeling throughout the brain section. However, a weak residual signal persists as spots over the glioma tissue. Note absence of fluorescence in the section processed without TAMRA-FP, indicating no detectable autofluorescence in the Cy3-window used for TAMRA-FP imaging. Note that PMSF (1 mM), in contrast to AEBSF (1 mM), and IDFP (100 μM) both efficiently inhibit TAMRA-FP labeling throughout the brain sections, yet leaving some residual activity over the tumor. Note in particular that MAFP (10 μM) efficiently inhibits probe labeling throughout the healthy brain regions but only modestly inhibited labeling of TAMRA-FP hotspots in the tumor. However, partial MAFP-sensitivity of TAMRA-FP signal was evident in tumors regions showing moderate probe fluorescence. The scale bars represent 1 mm. Images were adjusted for brightness and contrast