Visualization and quantification of cathepsin K activity in the CNS of WT mice. Confocal laser scanning micrographs were taken from horizontal sections through cortex (A) and hippocampus (B) of WT mice as indicated. Sections were prepared from 0.5% PFA-fixed brain tissue after a 45-min reaction with the cathepsin K substrate Z-Gly-Pro-Arg-4MβNA in the presence of NSA as precipitating agent of released 4MβNA under oxygen-free conditions and at pH 6.2 (A, green signals, and B, white signals), and after counter-staining with Draq 5 (A, blue signals). Cathepsin K activity was detected in vesicles (A, arrows) of both neuronal (B, circle) and glial cells (B, arrows) in the different brain regions analyzed by enzyme histochemistry. Bars - 100 μm (A), 50 μm (B).Results of cathepsin K activity assays performed with brain tissue lysates of WT (C-F, open bars, n = 6) and Ctsk-/- mice (grey bars, n = 3). Cathepsin K activity as determined by cleavage of Abz-HPGGPQ-EDN2ph at pH 5.5 was detected in the cortex, striatum/mesencephalon, cerebellum, and hippocampus of WT animals. Specific cathepsin K activity levels were calculated by division of relative fluorescence units (RFU) by the protein content of the respective samples. Specificity of the assay was proven by the use of tissue from cathepsin K deficient mice. Cathepsin K activity was well detectable in all brain regions analyzed and was highest in the hippocampus (C-F, open bars). Mean values ± standard deviations are depicted; levels of significance are denoted as ** for p < 0.01; *** for p < 0.001.