Fitc albumin

Fluorescent tracer uptake in nephrocytes to evaluate nephrocyte function was performed as previously described (Hermle et al., 2017 (link)). Briefly, nephrocytes were dissected in PBS and incubated with FITC-albumin (#A9771, Sigma) for 30 s. After a fixation step of 5 min in 8% paraformaldehyde, cells were rinsed in PBS and exposed to Hoechst 33342 (1:1000, #H1399, Thermofisher) for 20 s and mounted in Roti-Mount (#HP19.1, Carl Roth). Cells were imaged using a Zeiss LSM 880 laser scanning microscope. Quantitation of fluorescent tracer uptake was performed with ImageJ software. The results are expressed as a ratio to a control experiment with flies carrying the (heterozygous) GAL4 transgene but no UAS that was performed in parallel.
The parallel recording of two fluorescent tracers of different size to study the passage of the slit diaphragm was carried out in the same way as the assay for nephrocyte function, except that nephrocytes were simultaneously incubated with tracers FITC-albumin (0.2 mg/ml) and Texas-Red-Dextran (#D1863, Thermofisher, 10 kDa, 0.2 mg/ml) for 30 s after dissection. Cells were imaged using a Zeiss LSM 880 laser scanning microscope. Image quantitation was performed with ImageJ software for each channel separately. Alternative tracers were Texas-Red-Avidin (66 kDa, #A2348, Sigma) and Alexa Fluor 488 wheat germ agglutinin (38 kDa, #W11261, Thermofisher).

We prepared the ex-vivo whole brain sample by preparing a mixture of gelatin (Sigma Aldritch, G2500) in PBS (ThermoFisher Scientific, $10\times$ pH 7.4, 70011069) diluted 1:10 at 10% weight-per-volume for a maximum amount of 10 ml for one animal. The mixture was placed on a hotplate and heated to 40°C to 45°C to keep the gelatin dissolved. Next a heparinized PBS mixture is prepared by adding 600 iu of heparin (ThermoFisher Scientific, H7482) to 30 ml of PBS and is kept at 40°C to 45°C. Next, 30 mg of fluorescein isothiocyanate (FITC)–albumin (ThermoFisher Scientific, A23015) is added to 1 ml of PBS. Just before perfusion, we filled a beaker with crushed ice and added the FITC–albumin solution to the gelatin solution, shaking gently. Next, we performed a cardiac perfusion with the heparinized saline followed by the FITC–albumin–gelatin. The mouse was put head down into the crushed ice for 15 min. Afterward, the brain was extracted, placed in 4% paraformaldehyde (Electron Microscope Sciences, Diluted 1:8, 15714S) for 6 h, then placed in PBS. To finish the fixation process, the brain in PBS was placed on a horizontal shaker for 3 days.

Pulmonary vascular permeability was quantitatively measured by extravasation of FITC-Albumin (Sigma-Aldrich, St. Louis, MO, USA) into lung parenchyma [42 (link)]. Briefly, after 22 hours of rI/R, FITC-Albumin 5mg/kg was injected via tail vein of mice under surgical anesthesia. After 2 hours injection, all mice received thoracotomy under terminal anesthesia with 1.5% isoflurane via mask, and their lungs were harvested after 10 ml PBS transfusion to pulmonary artery for washing intravascular blood. The isolated lungs were frozen in liquid nitrogen, then homogenized and centrifuged (3,000g, 4°C for 10 minutes). Supernatant (100μl) was collected and FITC-Albumin optical density, which indirectly reflects the permeability of pulmonary microvascular, was quantitated by Varioskan Flash multimode reader (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation and emission of 494 nm and 520 nm, respectively. The assessments were made by an investigator blinded to the experimental protocols.