Bx60 fluorescent light microscope

Paraffin-embedded sections from the formalin-fixed brain tissues were stained by with Nissl stain kit (IHCWORLD, Woodstock, MD) to detect Nissl body according to the manufacturer’s instruction. The sections were also used for immunofluorescent staining for p-tau S³⁹⁶. BX60 Olympus fluorescent/light microscope equipped with DP-70 camera (Olympus, Tokyo, Japan) was used for imaging. The intensities of fluorescence were quantified by Image-Pro Plus (MediaCybernetics, Bethesda, MD, USA).

hBMVECs were cultured on fibronectin-coated chamber slides (BD Bioscience). Following drug treatments, the cells were fixed with 4% (wt/vol) paraformaldehyde, permeabilized by the addition of 0.25% Triton X-100, and blocked by 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS). The slides were immunostained for phospho-MLC (Ser¹⁹) as well as stained with Phalloidin for F-actin (F-actin Visualization Biochem kit, Cytoskeleton, Inc, Cat#: BK005, Denver, CO) and DAPI for nucleus (4′,6-diamidino-2-phenylindole; ThermoFisher Scientific, Houston, TX). The cells were imaged by BX60 Olympus fluorescent/light microscope equipped with DP-70 digital camera (Olympus, Tokyo, Japan). The density of fluorescence was analyzed by ImageJ (NIH, Bethesda, MD).