Fluorescein isothiocyanate fitc dextran

The efficiency of glymphatic clearance was evaluated using in vivo two-photon imaging [7 (link)]. Briefly, mice (n = 6 per group, 3 male and 3 female) were anesthetized and a thin cranial window was created at the parietal. Fluorescein isothiocyanate (FITC)-dextran (70 kDa; Sigma-Aldrich, USA) was dissolved in artificial cerebrospinal fluid at a concentration of 1%; 10 μl of FITC was injected into the cisterna magna using a microsyringe connected with a syringe pump controller. 0.2 ml of 1% rhodamine B (Sigma-Aldrich, USA) in saline was injected intravenously to show the brain vascular before imaging. Two-photon imaging on the right parietal cortex (2 mm caudal from bregma, and 1.7 mm lateral from the midline) was performed using a two-photon laser scanning microscope (Leica, Germany) equipped with a water immersion objective (25×). To monitor the clearance of FITC-dextran injected into the brain parenchyma, three-dimensional (3D) xyz stacks (512 × 512 pixels, 2-μm resolution) were taken up to 300 μm below the cortical surface at 5, 15, 30, 45, and 60 min after the injection of the FITC-dextran, the overall fluorescence intensities were analyzed. Besides, images 100 μm below the cortical surface were obtained and the fluorescence intensities in the paravascular space were analyzed to examine the efficiency of glymphatic clearance.

All chemicals were used without additional
purification. Fluorescein isothiocyanate (FITC)-dextran, molecular
weight (Mw) 70 kDa, rhodamine B isothiocyanate (RITC), and 2-mercaptoethanol
were purchased from Sigma-Aldrich. Cetyltrimethylammonium bromide
(CTAB, 99+%), tetraethyl orthosilicate (TEOS, 98%), and ammonium hydroxide
(NH₄OH, 28–30 wt %) were purchased from Acros. 2-[Methoxy(polyethyleneoxy)6–9propyl]trimethoxysilane
(PEG-silane, Mw 459–591 g/mol), trimethoxysilylpropyl-N,N,N-trimethylammonium
chloride (TA-silane, 50% in methanol) and (3-trihydroxysilyl)propylmethylphosphonate
(THPMP-silane, 42% in water) were acquired from Gelest. Dulbecco’s
Modified Eagle Medium (DMEM) was purchased from Gibco Co. Fetal bovine
serum (FBS) were purchased from HyClone, GE. An anti-CD31 antibody
(BS1574) was purchased from Bioworld, an anti-CD140b antibody (16–1402–82)
was purchased from Invitrogen, and an anti-ZO-1 antibody (ab221547)
was purchased from Abcam. Secondary goat antirabbit IgG-FAM 488 (TAFB02-F)
was purchased from BioTnA. Ethanol at 99.5% was purchased from Choneye
Pure Chemicals. Doxorubicin (DOX) was obtained from Scinopharm Taiwan
Ltd.

Sterile culture ware was obtained from Fisher Scientific (Pittsburgh, PA, USA), reagents and chemicals were purchased from Sigma-Aldrich or Bio-rad laboratories (Hercules, CA, USA), while Mini-Protean^® TGX™ gels 4–15% (#456-1084) from Bio-rad laboratories was used for gel electrophoresis. Dextran-Cascade Blue^® (10,000 MW; #D-1976) was obtained from Life Technologies, while Fluorescein isothiocyanate (FITC)-dextran (3,000–5,000 MW; #FD4) and Rhodamine B isothiocyanate (RITC)—dextran (70,000 MW; #R9379) were purchased from Sigma-Aldrich.

Dichlorodihydrofluorescein diacetate (DCFH-DA), dihydroethidium (DHE), lipopolysaccharide (LPS), and fluorescein-isothiocyanate- (FITC-) dextran (average MW 3,000-5,000) were purchased from Sigma-Aldrich (California, USA). Naphthalene-2,3-dicarboxal-dehyde (NDA) was obtained from Life Technologies (Carlsbad, CA, USA). SYBR Green PCR Master Mix was purchased from Roche (Basel, Switzerland). All other reagents used were of analytical grade.

The insert well was coated with collagen in an incubator for 30 min. One million cells were cultured in a 24-well transwell plate (Corning Life Sciences, Lowell, MA, USA) for 72 h until monolayer formation. The endothelial electrical resistance of the cells was measured using a Millipore Millicell ERS-2 system (Millipore Corporation, Billerica, MA, USA). The electrical resistance was calculated from the background-corrected values. The permeability assay was performed using Fluorescein isothiocyanate (FITC)-dextran (Sigma, St. Louis, MO, USA). Basal medium was added to the bottom well, and 1 mg/mL FITC-dextran was added to the upper well. After 15 min of incubation, FITC-dextran (excitation maximum 485 nm; emission maximum 530 nm) was measured with the media of the bottom well using a spectrophotometer (Multiskan FC, Thermo Fisher Scientific, Waltham, MA, USA).