PTH treatment protocol

Fifty-four animals (27 control and 27 knock out) were randomly assigned to treatment with single daily injections of either vehicle (10 mM acetic acid containing 2% heat-inactivated mouse serum) or 80 ng/g BW of h(1–34)PTH for 29 days as previously reported [22]. There were four treatment groups (vehicle/KO: 13 animals, PTH/KO: 14 animals, vehicle/control: 13 animals, PTH/control: 14 animals). Bone density was measured by DXA before and after 29 days of treatment.

Three-dimensional histology using two-photon microscopy was performed using previously published methodology [25]. Femurs were cleaned of soft tissue, fixed in 4% PFA, rehydrated in 30% sucrose at 4°C and placed in Tissue Tek optimum cutting temperature compound (Sakura Inc., Torrance, CA). Samples were immediately frozen in EtOH and dry ice and stored at -80°C. Femur bone and marrow tissue were cut longitudinally with a cryostat to expose bone marrow cells and bone (~250μm) for 2-photon microscopy. 2-photon microscopy images were acquired using an Olympus BX61WI fluorescence microscope with a 20x, 0.95NA water immersion Olympus objective and dedicated single-beam LaVision TriM laser-scanning microscope (LaVision, Biotec), controlled by Imspector software. The metaphyseal region along the growth plate and individual trabeculae in the metaphysis region were scanned with femtosecond Chameleon Vision II titanium:sapphire (Coherent, Santa Clara, CA) laser pulses at 900 nm wavelength. The total imaging volume was 500x500x100 μm. At least 10 3-D images were analyzed per femur using Imaris software (Bitplane Inc, South Windsor, CT). Osteoclasts were visualized by GFP fluorescence intensity and anatomical position adjacent to bone as detected by second harmonic excitation. Quantification of osteoclast volumes was performed using Imaris (Bitplane Inc, South Windsor, CT) software, using the “surfaces” module to calculate osteoclast volume based on GFP fluorescence intensity.