Visualizing Albumin Leakage and Th17 Cells in Brain

FITC-labeled albumin (Sigma-Aldrich, A9771) was dissolved in buffered saline (10 mg/ml), and the fluorescent dye solution was slowly infused into the tail vein (10 ml/kg) as previously described^{63 (link)}. With a three-minute interval after the infusion completion, the mice were killed by decapitation and their brains were fixed for 48 h in 4% paraformaldehyde in PBS. Brain was cut on a vibratome (30 μm). Leakage of FITC-labeled albumin into brain parenchyma was observed under a fluorescent microscope. These sections additionally immunoreacted with anti-RORγ antibody (1:100, Rabbit, Abcam, ab207082) determination of Th17 cells in the perivascular and parenchyma of cortex and hippocampus. Cell counts were obtained using MetaMorph software (Molecular Devices). Cell numbers per brain region were divided by the respective tissue area and represented as cells/mm².