3 Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos 51 with an aqueous solution of 7% uranyl acetate for 20 minutes, followed by Reynold’s lead citrate for 10 minutes. Specimens were imaged at an acceleration voltage of 120 kV using a Tecnai 12 BioTWIN transmission electron microscope (FEI, Eindhoven, The Netherlands), equipped with a FEI 4k Eagle CCD camera. Virtual slides of zebrafish glomeruli were recorded at 18500x magnification, corresponding to a 1.2 nm pixel size at the specimen level, using automated data acquisition and stitching software.(35) Loss of anionic sites in the GBM The number of anionic sites in the GBM was measured using polyethylenimine (PEI) staining as described previously.(36, 37) Specifically, we measured the number of PEI particles per µm of GBM. PEI is a cationic polymer that binds to anionic sites. It was used a surrogate marker for HS-GAG, as HS-GAG are highly sulfated and therefore negatively charged. PEI is not specific to HS-GAG, as it binds to all anionic sites. Two samples each of homozygous dak/ext2 and WT embryos were incubated in 1% PEI (PEI-600, Polysciences, Warrington, PA) in 0.1 M sodium cacodylate buffer (pH 7.4) for 6 hours with constant agitation. The samples were washed after incubation and subsequently fixed with 2% phosphotungstic acid and 1% glutaraldehyde. The samples were then stained with 1% osmium tetroxide and embedded in epon LX-112. A selection of representative images containing at least 13 µm of GBM was analyzed using transmission electron microscopy. Foot process width Foot process width was analyzed using a selection of representative EM images obtained from three different homozygous dak/ext2 mutants and three different WT embryos. All images contained at least 9 µm of contiguous GBM. The formula was used to calculate foot process width.(38) Assessing glomerular permeability The quantitative dextran tracer injection method (adapted from the qualitative method described by Ebarasi et al.(39)) was used to measure glomerular permeability. This method is not used for an assessment of charge- or size-selectivity, but for analyzing functionally significant alterations to the global permeability of the GFB. A total of 32 homozygous dak/ext2 and 35 WT embryos were used for four experiments as follows: at 5 dpf, the zebrafish embryos were anesthetized with 4% tricaine methanesulfonate (4 mg/ml), injected intravenously with 1 nl of a mixture containing FITC-labeled lysinefixable 70-kDa dextran (25 mg/ml; Invitrogen, Waltham, MA) and TRITC-labeled 3-kDa dextran (100 mg/ml; Invitrogen). As a positive control, WT zebrafish embryos
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