γh2ax

Animals were euthanized and perfused with saline solution 4 h (for inflammation and DNA DSB staining) or 3 days (for cell proliferation and vasculature staining) after ²¹²Pb-αVCAM-1 treatment. Inflammation, DNA DSBs, tumor cell proliferation, pericytes, microglia, and astrocytes were evaluated using primary antibodies against VCAM-1 (5 µg/mL, SoutherBiotech), γH2AX (2 µg/mL, Abcam), Ki67 (0.35 µg/mL, Dako), CD31 (5 µg/mL, PB Bioscience), platelet derived growth factor receptor beta (PDGFRβ) (2 µg/mL, Santa-Cruz), CD68 (1 µg/mL, Merck Millipore), and glial fibrillary acidic protein (GFAP) (3 µg/mL, Dako), respectively. Immunostaining protocols were performed as previously described.^{13 (link)} Tissue sections were examined at 20x magnification for VCAM-1 and at 40x for Ki67, γH2AX, CD31, PDGFRβ, CD68, and GFAP using a Leica DMi8 microscope. BM were identified by Hoechst 33342 counterstaining and through green fluorescent protein expression of MDA-231-Br cells. Whole brain images were obtained using Metavue software. For quantification of VCAM-1, CD31, γH2AX, and Ki67, three slices per animal were used and vessels, foci, and nuclei were automatically counted (ImageJ). Quantitative results of γH2AX and Ki67 staining are expressed as the percentage area of biomarker expression relative to the total tumor area. Vessel diameter was quantified as previously described.^{16 (link)}

After treatment, cells were fixed with 4% formaldehyde for 20 min at room temperature. Cells were permeabilized by Triton X-100 and subjected to goat serum blocking (Beyotime Biotechnology, Haimen, China). Cells were then incubated with primary antibodies against γ-H2AX (1:100 dilution) (Santa Cruz, CA, USA) and β-catenin (1:200 dilution) (Beyotime Biotechnology, Haimen, China) overnight at 4 °C. Cells were rinsed with PBS twice and then incubated in the dark with respective secondary antibodies for 60 min and DAPI for 5 min (Beyotime Biotechnology, Haimen, China). Using a fluorescence microscope (Leica, Weltzlar, Germany), the number of γ-H2AX foci per cell was quantified. Then, β-catenin fluorescence was measured by laser confocal microscopy (Olympus, Tokyo, Japan).