Cathepsin K detection in mouse brain parenchyma. Messenger RNA was isolated from tissue extracts prepared from different brain regions of WT or Ctsk-/- mice. RT-PCR reactions with the primer pairs cathepsin K 5'-GCC AGG ATG AAA GTT GTA TG-3' (forward) and 5'-CAG GCG TTG TTC TTA TTC C-3' (reverse) as well as β-actin 5'-GCC AGG ATG AAA GTT GTA TG-3' (forward) and 5'-CAG GCG TTG TTC TTA TTC C-3' (reverse) resulted in the amplification of cDNAs of the expected lenghts of approximately 358 bps for cathepsin K specific mRNA (Cath K) and 335 bps for the loading control (β-actin) as revealed by agarose gel (1,5%) electrophoresis. In negative controls, cDNA template was omitted (Co). Markers of fragment sizes are given in lanes labeled with St. (A) Cathepsin K mRNA was detected by RT-PCR in cerebral cortex tissue from WT mice (n = 3), but not in samples obtained from Ctsk-/- mice (n = 3; upper panel). (B) Cathepsin K mRNA was detected in all four investigated brain regions in WT animals. Bps - base pairs; St - standard DNA ladder; Co - negative RT-PCR control without template; H - hippocampus; Str - striatum/mesencephalon; Cb - cerebellum; Cx - cortex. Confocal laser scanning micrographs were taken from horizontal sections through choroid plexus (C), cerebellum (D), cortex (E), and hippocampus (F) of WT or Ctsk-/- mice as indicated. Sections were prepared from PFA-fixed brain tissue, immunostained for cathepsin K (green signals, lower panels - middle), counter-stained for nuclear DNA with Draq 5 (red signals, lower panels - right), and inspected with fluorescence and phase contrast (lower panels - left) microscopy. Cathepsin K protein was immunodetected in ependymal cells (C, arrows), neurons (F, circles) and glial cells (F, arrows) of WT mice (C and F) but not in negative antibody controls (E) or in sections prepared from Ctsk-/- brain tissue (D), indicating specificity of cathepsin K immunolabeling. Bars - 100 μm (C-F). CP - choroid plexus; Cb - cerebellum; Cx - cortex; H - hippocampus.
Dauth et al. BMC Neuroscience 2011 12:74 doi:10.1186/1471-2202-12-74