Histological Analysis of Mouse Bone and Vasculature

Long bones were decalcified in 10% EDTA, embedded in paraffin and sections were stained with hematoxylin, eosin (H&E) and orange G. All mouse brains were stained with H&E. For IHC, slides were autoclaved at 121°C for 10 min in target retrieval solution (DAKO, Santa Clara, CA, USA), followed by CD34 antibody (1:2 000, Abcam). Tartrate-resistant acid phosphatase (TRAP) staining was performed as previously described (18 (link)). For histomorphometric analysis, bright-field microscopic images were acquired randomly with a AxioScan Z1 microscope using a 20X/NA 0.8 plan apochromat objective (Zeiss, Oberkochen, Germany). CD34-stained blood vessel areas were quantified using ImageJ (NIH, Bethesda, MD, USA) as described previously (34 (link)). TRAP⁺ osteoclast number per millimeter of tumor/bone interface was counted at 20X magnification as described previously (34 (link), 54 (link)). All the analysis was performed blindly.