Experimental animals were repeatedly immobilized in a perspex tube on a custom-made stage (Effenberger, Munich, Germany) for intravital microscopy at three time points after PPE implantation (i.e. days 3, 7 and 14, see Supplemental Fig. 1). The mice remained awake during intravital microscopy and were constantly monitored for signs of discomfort or distress. A modified Zeiss microscope (Axiotech Vario; Zeiss, Göttingen, Germany) was used for imaging. Fluorescein isothiocyanate (FITC)-labeled dextran (Sigma, Deisenhofen, Germany; MW 500,000; 0.05–0.1 mL of a 5% solution in 0.9% saline) was used to stain blood plasma for the visualization of the vascular network and rhodamine 6G (Molecular Probes, Eugene, OR; 0.04 mL of a 0.05% solution in 0.9% saline) was used to stain leukocytes for the analysis of leukocyte-endothelial cell interactions (Fig. 1B, C). The fluorescent dyes were administered via tail vein injection. For selective observation of FITC-labeled plasma and rhodamine 6G-labeled leukocytes epi-illumination with a 100 W mercury lamp with selective filter blocks (Zeiss, Göttingen, Germany) was used. Both renally eliminated fluorescent markers are well-established for in vivo labelling and feature a blood half-life sufficient for in-vivo imaging over a time period of at least 2 h [19 (link), 20 (link)].
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