Mouse antihuman cathepsin k

The free-floating sections in 12-well plates were incubated with 5% normal goat serum to block the nonspecific immunoglobulin binding sites of the secondary antibodies. The sections were incubated first with antibodies against mouse antihuman CD68 (eBioscience) and mouse antihuman cathepsin K (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C. Then, the sections were incubated with DyLight^TM 488-conjugated goat anti-mouse IgG (Thermo Fischer Scientific, Waltham, MA, USA) for 1 h. The sections were then incubated with rabbit polyclonal antihuman α₁-subunit (1:1000) [38 (link)] and β₁-subunit (1:1000) [39 (link)] antibodies overnight at 4 °C in the dark. Thereafter, the sections were incubated for 1 h with the DyLight^TM 550-conjugated goat anti-rabbit IgG (Thermo Fischer Scientific). To show the cell nuclei, sections were stained with Hoechst 33342 and DRAQ5 (Cell Signaling Technology, Frankfurt am Main, Germany) for immunofluorescence microscopy and LSM confocal microscopy, respectively, for 15 min. The sections were coverslipped with Aqua Poly/Mount (Polysciences Inc., Warrington, PA, USA) and analyzed with an LSM510 confocal microscope (Carl Zeiss, Jena, Germany) [50 (link)].

The free-floating sections in 12-well plates were first treated with 0.3% H₂O₂ in 0.05 M Tris-buffered saline (TBS) for 20 min. The sections were incubated with 0.25% Triton X-100 in 0.6 M TBS, pH 7.6, for 30 min. The nonspecific immunoglobulin binding sites were blocked by incubation of the sections with a blocking solution containing 5% normal goat serum (Vector, Burlingame, CA, USA) and 2% bovine serum albumin (Sigma-Aldrich, Taufkirchen, Germany). The consecutive sections were incubated with mouse antihuman cathepsin K (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse antihuman MCT (Santa Cruz Technology, Santa Cruz, CA, USA), mouse antihuman CD68 (eBioscience, San Diego, CA, USA), mouse antihuman HL-DR (eBioscience, San Diego, CA, USA), and polyclonal rabbit antihuman α₁- (1:1000) [38 (link)] and β₁-subunits (1:1000) [39 (link)] antibodies at 4 °C. The sections were incubated with biotinylated goat anti-mouse or goat anti-rabbit IgG (1:500) (Vector) for 1 h, respectively, and subsequently with avidin-biotin peroxidase complex (1:100) (Vector) for 1 h. The immunohistochemical reaction was developed in all sections with 0.05% 3,3’-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Taufkirchen, Germany) in 0.05 M Tris-HCl buffer, pH 7.6, containing 0.01% H₂O₂ and 0.01% nickel sulfate for 15 min [38 (link),50 (link)].