Biotinylated goat anti rabbit secondary antibody

Mouse brain was dissected and post-fixed with 4% formaldehyde at 4°C for 16 hr; tissues were sectioned with Vibratome 1000 Plus (Rankin Biomedical, Holly, MI) at 100 μm thickness and incubated with antibody in free-floating method. For ABC-DAB-Nickel staining, tissue sections were first bleached in 1 x TBS containing 0.03% H₂O₂ for 30 min, and then blocked in TBST (TBS +0.05% Triton X-100) containing 5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) and 5% normal goat serum (NGS from Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 60 min, and incubated with Rabbit polyclonal Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) primary antibody [(1:500) #9101, Cell Signaling Technology, Danvers, MA, USA] diluted in blocking solution overnight at 4°C. Sections were then washed three times with TBST and incubated with secondary biotinylated goat-anti-rabbit antibodies (1:1000, Vector Laboratories, Burlingame, CA, USA) for 1 hr at room temperature. After three TBST washes, sections were incubated in the Avidin-Biotin pre-mix solution (1:200, Vector Laboratories, Burlingame, CA, USA). After 3 1xTBS washes, positive immunoreactivity signals were visualized using a Nickel-DAB method [DAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3’-diaminobenzidine SK-4100, Vector Laboratories, Burlingame, CA, USA or Sigma-Aldrich, St. Louis, MO, USA].

For histological analysis, femurs were excised, the needle was removed, and the sample was fixed in 10% neutral buffered formalin for at least 24 h, and decalcified in 10% EDTA, pH 7.5 for 5–8 days at room temperature. The samples were embedded in paraffin along their long axis and sectioned (5 μm). Before staining, slides were deparaffinized and gradually rehydrated though series of ethanol washes. Sections were stained for immunohistochemistry (IHC) and tartrate-resistant acid phosphatase (TRAP). For IHC, in the antigen-retrieval step, slides were incubated in pre-boiled citrate buffer (#005001, Life Technologies, Grand Island, NY, USA) for 10 min. Endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 30 min. The slides were then incubated overnight with primary rabbit anti-β-catenin antibody (#9562, Cell Signaling, Danvers, MA, USA) diluted 1:200. The secondary biotinylated goat anti-rabbit antibody (#BA-1000, Vector Laboratories, Burlingame, CA, USA) diluted 1:200 was used with the Vectastain avidin biotinylated enzyme complex system for visualization. TRAP staining for osteoclasts was performed using a Leukocyte Acid Phosphatase Kit (#387A, Sigma-Aldrich) and counterstained with hematoxylin.