Paraffin-embedded tumors from the IgG1 control-treated (n = 6) and hSFRP2 mAb-treated (n = 6) metastatic osteosarcoma mice were sectioned at 6 µm, and immunohistochemistry was performed as described previously (34). Briefly, slides were deparaffinized and rehydrated using Discovery wash (950-510, Roche Tissue diagnostics, Indianapolis, IN, USA). Antigen retrieval was performed using EDTA buffer solution for 32 min on the Discovery Ultra staining platform. Endogenous peroxidase was blocked with Discovery Inhibitor solution (760-4840, Roche Tissue Diagnostics), and the tumors were then incubated with CD31 antibody (1:200, #ab28364, Abcam) for 1 h RT. A negative control with no primary antibody added was generated to ensure the specificity of the staining. Slides were then incubated with an HRP-conjugated OmniMap anti-rabbit secondary antibody (1:200, #760-4311, Ventana Medical System, Tucson, AZ, USA) for 20 min at 37 °C, and then precipitated with a DAB substrate following chromomap DAB kit protocol instructions (#760-159, Ventana Medical Systems). Five fields per tissue slice were analyzed from pictures taken at 40× using the EVOS FLc Digital Imaging System (Thermo Fisher Scientific), and hot spots of positively stained cells were counted within each field as previously published [16 (link)]. The numbers obtained for all 5 fields were then averaged.
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