For immunohistochemical and immunofluorescent staining, mice and rats were rapidly euthanized in CO2 and transcardially perfused with phosphate-buffered saline (PBS) followed by 4% neutral buffered paraformaldehyde (Fisher Scientific). Brains were extracted, equilibrated in 30% sucrose, sectioned coronally on a cryostat (Leica Biosystems) at 30 μm and collected and rinsed in 0.1 M TBS. Mouse brains for vessel length measurement by the FITC-albumin infusion approach were prepared by emulsion fixation with 4% paraformaldehyde after receiving FITC-albumin (Sigma-Aldrich, St. Louis, MO, USA) infusion, sucrose equilibration and sectioning.
Fitc albumin
Manufactured by Merck Group
Sourced in United States, Germany
FITC-albumin is a fluorescently labeled protein commonly used as a tracer in various research applications. It is composed of bovine serum albumin (BSA) conjugated with the fluorescent dye fluorescein isothiocyanate (FITC). The FITC labeling allows for the visualization and tracking of the albumin molecule in experimental settings.
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Lab products found in correlation
Texas red dextran, by Thermo Fisher Scientific (3 mentions)
Cryostat, by Leica (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Roti mount, by Carl Roth (2 mentions)
Fitc albumin, by Thermo Fisher Scientific (2 mentions)
Fitc dextran, by Merck Group (2 mentions)
Tritc dextran, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
2480 wizard2, by PerkinElmer (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Spark multimode microplate reader, by Tecan (1 mentions)
D12331, by Research Diets (1 mentions)
Cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
Tri carb 2910tr liquid, by PerkinElmer (1 mentions)
64 protocols using fitc albumin
1
Immunohistochemical Tissue Preparation Protocol
2
Quantifying ANP-2xGFP Accumulation and FITC-Albumin Endocytosis in Drosophila Nephrocytes
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For the ANP-2xGFP accumulation assay, garland nephrocytes from wandering third instar larvae were microdissected in HL3.1 saline [37 (link)], fixed in 4% PFA in PBS for 10 min, stained with DAPI for 20 min, washed with PBS, and mounted in Mowiol. ANP-2xGFP accumulation per nephrocyte area (CTCF = Corrected Total Cell Fluorescence) was analyzed and quantified with ImageJ after subtracting the autofluorescent background of dissected larvae. For each genotype, at least 75 nephrocytes of 10 independent larvae were quantified.
For FITC-Albumin endocytosis assays, garland nephrocytes were dissected as described above and incubated with 1 mg/ml FITC-Albumin (SIGMA #A9771) in HL3.1 saline for 1 min. After two washes with PBS, cells were fixed and processed as described above. For each genotype, at least 15 independent larvae (> 50 nephrocytes total) were quantified.
For FITC-Albumin endocytosis assays, garland nephrocytes were dissected as described above and incubated with 1 mg/ml FITC-Albumin (SIGMA #A9771) in HL3.1 saline for 1 min. After two washes with PBS, cells were fixed and processed as described above. For each genotype, at least 15 independent larvae (> 50 nephrocytes total) were quantified.
3
Cellular Uptake Kinetics of Fluorescent and Radiolabeled Compounds
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The accumulation studies of fluorescein isothiocyanate-labelled albumin (FITC-albumin, Merck) were performed in 24-well plates (105 cells per well). The cells were incubated with 24 µg/mL FITC-albumin, and Passive Lysis Buffer (Promega) was added to lyse the cells after 2 h treatment. The accumulated FITC-albumin was quantified by measuring the fluorescence intensity of cell lysates (Tecan Spark® multimode microplate reader). Fluorescence intensity was plotted against total protein concentration determined by the commercially available Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific).
The accumulation studies of 15-mers were performed in 24-well plates (105 cells per well). The cells were treated with either 20 µg/mL 68Ga-radiolabeled 15-mer or 27 µg/mL 99mTc-radiolabeled 15-mer for 2 h. The accumulated radioactivity was determined using an automatic γ counter (2480 Wizard2, PerkinElmer), with results presented as counts per minute related to the amount of cell protein in each sample.
The accumulation studies of 15-mers were performed in 24-well plates (105 cells per well). The cells were treated with either 20 µg/mL 68Ga-radiolabeled 15-mer or 27 µg/mL 99mTc-radiolabeled 15-mer for 2 h. The accumulated radioactivity was determined using an automatic γ counter (2480 Wizard2, PerkinElmer), with results presented as counts per minute related to the amount of cell protein in each sample.
4
Hypertension-Induced Hypothalamic Vascular Alterations
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Rat studies were approved by the Animal Ethics Committee of the government of Universidad Autónoma de Madrid (Spain). Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats (Envigo; Indianapolis; USA) were group housed with continuous free access to pelleted food and water and were maintained at 23°C with constant humidity on a 12-h light–dark cycle. All rats used in experiments were directly purchased (Envigo), displayed good general health and immediately entered experiments upon sufficient acclimatization to the facility. Hypothalamic vascular profiles were assessed in 16-week-old male WKY rats either fed 4 weeks HFHS diet (D12331; 58% of calories from lipids; Research Diets, New Brunswick, NJ) or SC diet and compared to littermates with spontaneously hypertensive rats (SHR) maintained on SC diet. Bodyweight was assessed repeatedly as well as blood pressure using tail cuff measurements conducted over the course of four consecutive days (Niprem 645 blood pressure system; Cibertec, Madrid, Spain). At the day of sacrifice, rats received 10 ml/kg of FITC-albumin (10 mg/ml in 0.9% NaCl; MW 69 kDa, Merck, Germany) via the tail vein. After circulation of the tracer for 5 min, rats were decapitated, blood was collected for determining circulating leptin levels and brains were rapidly immersion-fixed in 4% PFA in 0.1 M PBS (pH 7.4) for 72 h at 4°C.
5
Compound Uptake Assay Protocol
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To assess compound uptake, several compounds were tested for specific receptors. FITC-Albumin (Sigma-Aldrich, St. Louis, MO, USA) was prepared at a stock of 500 mg/mL and diluted in Krebs buffer as appropriate. Uptake assay was run for 90 min at 37 °C, before cells were lysed using Cell Lysis Buffer (Invitrogen, Waltham, MA, USA) and fluorescence measured using ClarioStar Plus (BMG Labtech GmbH, Ortenberg, Germany) to determine compound uptake. For radiolabeled compound studies, 14C-Creatinine and 3H-Mannitol were prepared to a stock of 10 μM and dosed in 200 μL (upper chamber) or 800 μL (lower chamber) of Krebs buffer on the appropriate side to determine uptake of the compound. Uptake assay was run for 90 min at 37 °C before reaction was stopped at 90 min with ice-cold Krebs and membranes cut out of inserts and placed in scintillation vials immersed in Ultima GOLD (Perkin Elmer, Waltham, MA, USA) scintillation fluid and DPM measured using a Tri-Carb 2910TR liquid scintillation counter (Perkin Elmer, Waltham, MA, USA).
6
Fluorescent Tracer Uptake in Nephrocytes
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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).
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).
7
Quantitative Proteomic Analysis with ICAT
8
Lung Permeability Assessment in LPS-Induced Mice
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Six mice were divided into two groups, which included the control and model groups. The model group (n = 3) was instilled i.n. with 0.5 mg of LPS/kg b.w. The control group (n = 3) was instilled i.n. with 50 µl of PBS. Five hours after the LPS or PBS instillation, 5 mg of FITC-Albumin/kg b.w. (Catalog number: A9771; Sigma-Aldrich, St. Louis, MO, USA) was instilled i.n. into the airway of the model and control groups. The FI of the plasma FITC-Albumin was measured as described above.
9
Visualizing Intestinal Capillary Leakage
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Using fluorescein isothiocyanate (FITC)-albumin as a plasma marker, capillary leakage of the intestine was observed visually. Fifteen minutes before the rats were sacrificed 1 mg/0.5 mL FITC-albumin (Sigma, MO, USA) was injected into the femoral vein. After 15 min, the abdomen was opened and the intestinal tissues and small intestinal mesentery were harvested. The intestinal tissues were fixed in 4% paraformaldehyde, and embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Inc.). Frozen sections (10-μm thickness) were cut at −20 °C with a cryomicrotome (Reichert Jung 2800, Leica, Germany). The small intestinal mesentery was placed on glass slides. They were observed under a confocal microscope and pictures were taken.
10
Fluorescent Albumin-Based Brain Microvasculature Imaging
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