X-AC9

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Four CypA WT and two knockout (KO) 13-week-old adult mice were euthanized and fixed in 10% formalin. Femurs were isolated and embedded in paraffin. Paraffin sections (5 μm thick) were dewaxed in xylene and rehydrated in ethanol baths. Endogenous peroxidases were blocked by incubating the sections in 3% hydrogen peroxide for 20 min at room temperature. The sections were incubated with anti-Col II (II-II6B3-c; Development Studies Hybridoma Bank), anti-Col X (X-AC9), anti-p65 (sc-109; Santa Cruz Biotechnology, Santa Cruz, CA), or anti-CypA (sc-133494; Santa Cruz Biotechnology) primary antibody at room temperature for 1 h and overnight at 4°C and then incubated with a biotinylated secondary antibody (Dako, Carpinteria, CA) for 1 h at room temperature. Positive immune reactivity was detected using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and AEC chromogenic substrate (Dako) according to the manufacturer's instructions. A negative control was generated by replacing primary antibody solutions with phosphate-buffered saline (PBS). The sections were counterstained with hematoxylin for 30 s and then rinsed for 5 min in running water. Photomicrographs were acquired using an Olympus BX51 microscope and a MicroFire digital microscope camera with PictureFrame software.

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