Goat anti cd31 antibody

Whole-mount staining was performed according to our previous reports [37 (link),38 (link)]. Tissues were cut into thin slices and fixed in 4% PFA overnight, and then exposed to 20 μg/mL proteinase K. Thereafter, the tissues were incubated with goat anti-CD31 antibody (R&D System Inc, Cat. No. AF-3628) overnight at 4 °C, which was followed by staining with a donkey anti-goat secondary antibody (Abcam, Tokyo, Japan) for 2 hours at room temperature. Slides were mounted and examined under a microscope (Keyence, Osaka, Japan). We scanned 5 thin sections at 3-μm distances of each sample and projected three-dimensional images of each tissue sample. Quantitative analyses from at least six different sections were performed using the Adobe Photoshop CS software program (Adobe systems, Tokyo, Japan).

Whole-mount staining was performed as described previously.^{32 ,33} Briefly, tissues were cut into thin slices, fixed in 4% paraformaldehyde overnight, and then exposed to 20 μg/mL proteinase K. Thereafter, the tissues were incubated with a goat anti-CD31 antibody (R&D Systems Inc., Minneapolis, MN, USA, Cat. No. AF-3628) overnight at 4°C, followed by staining with a donkey anti-goat secondary antibody (Abcam, Tokyo, Japan) for 2 h at room temperature. To assess vascular permeability and leakiness, lysine-fixable tetramethyl rhodamine dextran with a molecular weight of 70 kDa or 2000 kDa (Invitrogen) was injected into the tail vein. The mice were sacrificed five minutes after injection with 2000 kD and 15 min after injection with 70 kD. The slides were mounted and examined under a microscope (Keyence Corporation, Osaka, Japan). We scanned five thin sections at 3-μm distances from each sample and projected three-dimensional images of each tissue sample. Quantitative analyses of at least six different sections were performed using the Adobe Photoshop CS software (Adobe Systems, Tokyo, Japan).