Chapter 2 40 cacodylate buffer, dehydrated sequentially in 70%, 80%, 90%, and 100% ethanol, and then immersed in 1:1 propylene oxide: epon LX-112 solution for 1 hour. After washing, infiltration with pure epon for 2 hours, and embedding in epon LX-112, samples were polymerized at 60°C for 2 days. Ultrathin (100 nm) sections were cut using a Leica EM UC6 ultramicrotome, collected on a slot grid, post-stained with 7% uranyl acetate for 20 minutes followed by Reynolds' lead citrate for 10 minutes, and examined at 120 kV in a JEOL JEM-1011 electron microscope equipped with a MegaView III digital camera (JEOL, Inc., Peabody, MA, USA). Evaluation of glomerular and tubular proteinuria Evaluation of proteinuria was performed by injecting dextran tracers intravenously and assessing both loss of fluorescence in the retinal vascular bed and resorption droplets. The different assessment models were implemented in different groups of zebrafish in separate experiments. ctns‒/‒ and wt zebrafish larvae (72 hpf) were anesthetized with 80 µg/ml tricaine methanesulfonate, then injected sequentially into the cardiac venous sinus (sinus venosus) with 2 nl of 50 mg/ml 70-kDa rhodamine B isothiocyanate-Dextran or 4.4-kDa tetramethyl rhodamine isothiocyanate-Dextran (Sigma). Injections were performed using FemtoJet micro-injector (Eppendorf, Hamburg, Germany). Success of injection was evaluated directly after injection with the Zeiss inverted fluorescence microscope. in the retinal bed was evaluated in ctns‒/‒ and wt zebrafish larvae injected with the 70-kDa tracer (n=20 for each genotype) by taking images after injection and 24h post injection. The peak fluorescence intensities in zebrafish retinal vascular bed were evaluated using fixed diameter circles by the ImageJ software16. Assessment of glomerular permeability and tubular reabsorption capacity were performed in separate groups of zebrafish injected with the 70-kDa or 4-kDa dextran tracers, respectively (n=10 in each group). Successfully injected larvae were fixed in 4% PFA at 60min post-injection traced separately for each larva. After 24h of fixation, they were transferred to 70% ethanol and kept at 4°C till processing. Larvae were washed with demineralized water, packed in groups of five using Shandon' Cytoblock' cell block preparation system, embedded in paraffin for oriented sectioning (3 µm). Sections containing PTECs were investigated by fluorescence microscope. Fluorescent droplets in the proximal tubules were counted, representing endosomes of dextran that have passed the glomerular filtration barrier and were subsequently reabsorbed by PTECs. Evaluation of glomerular filtration rate We evaluated GFR in zebrafish larvae using the fluorescein isothiocyanate (FITC)-inulin injection method as previously described36. Five percent (w/v) FITC-inulin (5-kDa)
RkJQdWJsaXNoZXIy MTk4NDMw