Biotinylated goat anti mouse or goat anti rabbit igg

Immunostaining was performed by the free-floating method as previously performed (44 (link)). For BrdU staining, the antigens were retrieved in the citric acid buffer (pH 6.0) at 95 °C for 15 min, then denatured in 2 N HCl for 30 min and neutralised by 0.1 M borate buffer (pH 8.5) for 15 min at room temperature. The sections were incubated overnight with mouse anti-BrdU antibody (1:1,000; Roche Life Science, Germany) and then incubated with the biotinylated goat anti-mouse IgG (1:200; Vector Laboratories, CA, USA) for 2 h at room temperature. For doublecortin (DCX) and Ki-67 staining, sections were incubated with mouse anti-DCX (1:200; Santa Cruz Biotechnology, TX, USA) or rabbit anti-Ki67 (1:1,000; Abcam, UK) antibodies, then secondary antibodies: biotinylated goat anti-mouse or goat anti-rabbit IgG (1:200; Vector Laboratories, CA, USA). Positive staining was visualised with the peroxidase method using the VECTASTAIN® ABC kit (HRP) (Vector Laboratories, CA, USA) and the DAB peroxidase substrate kit (Vector Laboratories, CA, USA).

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)].