Rhodamine 6g

EGTA, EDTA, HEPES, Ficoll 400, Sigma-Fluor^® High Performance LSC Cocktail, the analytical grade salts were purchased from Sigma (USA); L-[¹⁴C]glutamate and [³H]GABA (γ-[2,3-3H(N)]-aminobutyric acid) were from Perkin Elmer (Waltham, MA, USA). Rhodamine 6G were obtained from Molecular Probes (USA).

FeCl₂·4H₂O and FeCl₃·6H₂O were purchased from Sigma-Aldrich (Steinheim, Germany), sodium hypochlorite solution (NaOCl) from Bochemie (Bohumín, Czech Republic), and sodium citrate dihydrate from Lachema (Brno, Czech Republic). D-mannose was from Acros (Geel, Belgium). Ethylene glycol tetraacetic acid (EGTA) and HEPES were purchased from Sigma-Aldrich (USA). Acridine orange and rhodamine 6G were obtained from Molecular Probes (USA); Ficoll 400, L-[¹⁴C]glutamate, aqueous counting scintillant (ACS) were from Amersham (UK). Analytical grade salts were from Sigma-Aldrich (USA).

EGTA, EDTA, HEPES, Ficoll 400, aminooxiacetic acid, d-glucose, sucrose, Whatman GF/C filters, the fluorescent dye 2′,7′-dichlorofluorescein, Sigma-Fluor® High Performance LSC Cocktail, organic counting scintillant (OCS) and the analytical grade salts were purchased from Sigma (St. Louis, MO, USA); L-[¹⁴C(U)] glutamate, [³H]GABA (γ-[2,3-³H(N)]-aminobutyric acid) were from Perkin Elmer (Waltham, MA, USA). Rhodamine 6G and acridine orange were obtained from Molecular Probes (USA). JC-1, Mitochondrial Membrane Potential Assay Kit was from Novusbio.

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)].