Multicolor Immunohistochemistry of Tissues

Immunohistochemistry of whole-mount samples or tissue sections was performed as previously described (Kubota et al., 2009 (link)). The primary monoclonal antibodies used were hamster anti-CD31 (MAB1398Z; EMD Millipore), PDGFRα (14-1401; eBioscience), vascular endothelial cadherin (550548; BD), and F4/80 (MCA497R; Serotec). The primary polyclonal antibodies used were Alexa Fluor 488–conjugated anti-GFP (A21311; Molecular Probes), collagen IV (LSL-LB-1407; Cosmo Bio), anti-VEGF (sc-152; Santa Cruz Biotechnology, Inc.), and cleaved caspase-3 (9664; Cell Signaling Technology). The secondary antibodies used were Alexa Fluor 488 fluorescence–conjugated IgGs (Molecular Probes) or Cy3/Cy5 DyLight 549/DyLight 649–conjugated IgGs (Jackson ImmunoResearch Laboratories, Inc.). For nuclear staining, specimens were treated with DAPI (Molecular Probes). In some experiments, blood vessels and monocyte lineage cells were simultaneously visualized using biotinylated isolectin B4 (B-1205; Vector Laboratories) followed by fluorescent streptavidin conjugates (Molecular Probes).