Fitc albumin

Cellular endocytosis of albumin was investigated through application of different concentrations of FITC‐Albumin (Abcam) to cells on Matrigel‐coated beads. Matrigel‐coated beads for the assay were incubated with serum‐free medium with/without 50µg/ml, 100µg/ml, 500µg/ml, 1mg/ml FITC‐Albumin for 2 hours at 37°C. For immunofluorescence staining, cells were fixed using BD Cytofix, embedded into paraffin, sectioned and analyzed by Operetta high content imager and Columbus image analysis server (both PerkinElmer, Waltham, MA, US). For FACS analysis, cells were harvested from Matrigel‐coated beads using Trypsin/EDTA and albumin uptake was assessed using MACSQuant Analyzer and data analyzed with FlowJo software.

To detect BBB leakage in 3-NP-injected rats, experimental rats (n = 3 animals) were deeply anesthetized on days 3 and 7 after 3-NP injection and intravenously administered fluorescein isothiocyanate (FITC)-albumin (2 mg diluted in 0.1 ml saline; Sigma-Aldrich) via the tail vein. One hour after injection, animals were deeply anesthetized and perfused transcardially with fixative, as described above. Coronal cryostat sections (25-μm-thick) from animals injected with FITC-albumin were incubated overnight at 4°C with a mixture of rabbit polyclonal antibody against GRP78 (1:2000; Abcam) and one of following antibodies: goat polyclonal antibody against Iba1 (1:500; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad), or mouse monoclonal antibodies against GFAP (1:700; Millipore). This was followed by 2-h incubation with appropriate secondary antibodies as follows: Cy3-conjugated donkey anti-goat antibody (1:2000; Jackson ImmunoResearch), Cy3-conjugated goat anti-mouse antibody (1:2000; Jackson ImmunoResearch), or Alexa Fluor 647-tagged donkey anti-rabbit antibody (1:300; Thermo Fisher). Counterstaining of cell nuclei was carried out using DAPI for 10 min.