Goat anti cd31 primary antibody

To assess re-endothelialization, immunostaining of CD31 (an endothelial cell marker) was performed, as we previously reported,^{24 (link)} using a goat anti-CD31 primary antibody (R&D Systems; 1:150 dilution) and a biotinylated rabbit–antigoat secondary antibody (Vector Laboratories; 1:200 dilution). Staining of CD31 was visualized using streptavidin-HRP and 3,3-diaminobenzidine. For quantification, the luminal perimeter and the percentage of this perimeter that stained for CD31 on each section were measured using ImageJ software. The percentage of re-endothelialization was scored from 1–5 (1: < 20%, 2:20–40%, 3:40–60%, 4:60–80%, and 5:80–100%), as we previously reported.^{18 (link)}

Three days after intracarotid injections, mice were sacrificed and their brains were harvested and processed for frozen or paraffin sections, as previously described.^{16 (link)} Staining with H & E or DAPI (FluoroPure grade, Invitrogen) was performed to visualize the tumor. The GFP-labeled cells were visualized in frozen sections using fluorescence microscopy and in paraffin sections after deparaffinization and antigen retrieval with 1:1000 rabbit anti-GFP primary antibody (Novus) and 1:200 Donkey anti-rabbit green secondary antibody (Invitrogen). Detection of endothelial cells was performed with 1:250 goat anti-CD31 primary antibody (R&D) and 1:200 donkey anti-goat red secondary antibody (Invitrogen). For U87 xenografts, specimens were fixed in 10% formalin for 48 hours, placed in 30% sucrose until they sank, and then embedded in optimal cutting temperature medium and frozen.