Anti cd31 mab

Mice were briefly anaesthetized with 3% isoflurane and injected intrascrotally (i.s.) with IL-1β (50 ng), TNF (300 ng) or phosphate buffered saline (PBS) as vehicle control with or without fluorescent dye-conjugated anti-CD31 mAb (4 μg, clone 390, Thermo Fischer Scientific) to label endothelial cell (EC) junctions, in a 400 μl bolus for 2-4 h stimulation periods. For some intravital microscopy (IVM) experiments, cremasteric ischemia-reperfusion (IR) injury was induced as previously described (Colom et al., 2015 (link); Woodfin et al., 2011 (link)), with ischemia induced in the exteriorized cremaster muscle of anaesthetized mice by the placement of two non-crushing metal clamps (Interfocus, Schwartz Micro Serrefine) at the base of the exteriorized tissue for 40 min (mins). Subsequently, the clamps were removed and tissue reperfusion allowed. Control sham operated mice underwent surgical procedures without induction of IR. For other experiments, ischemia was induced in a non-surgical manner by the application of two orthodontic bands around the intact testes and scrotum to occlude the vasculature for 40 min, followed by 1-24 h of reperfusion.

Mice were anaesthetized by intramuscular injection (i.m.) of an anesthetic mix containing ketamine and xylazine in PBS and ears were injected intradermally (i.d.) with IL-1β (50 ng), or PBS as vehicle control, together with fluorescently labeled anti-CD31 mAb (4 μg, clone 390, Thermo Fischer Scientific) to label EC junctions, within a 40 μl bolus injection. Tissues were commonly analyzed 2-4 h later as detailed below.