A1r confocal laser scanning microscopy system

The mice were anaesthetized with 2.5% isoflurane (Abbott, North Chicago, IL) using a Univentor 400 anaesthesia unit (Univentor; Zetjun, Malta). The skulls of mice were partially cut open around the ROIs without damaging blood vessels. The mice were directly placed on a thermoplate (Tokai Hit, Tokyo, Japan), and a cover slip (Muto Pure Chemicals, Tokyo, Japan) was attached with proper pressure to flatten the brain surface. Images of the micelle crossing the BBB were acquired with an A1R confocal laser-scanning microscopy system (Nikon Corp., Tokyo, Japan) attached to an upright ECLIPSE FN1 (Nikon Corp.).

A172 cells were grown in MatTek glass-bottom dishes (with No. 1.5 coverslip), treated as indicated, washed with phosphate-buffered saline (PBS) supplemented with 1 mM CaCl₂ and 0.5 mM MgCl₂, then fixed using 4% paraformaldehyde in PBS. The cells were permeabilized with 0.2% Tween20 in PBS with Ca²⁺ and Mg²⁺, then blocked in antibody incubation buffer (1% bovine serum albumin in 0.2% Tween20 in PBS with Ca²⁺ and Mg²⁺). Cells were double-labeled overnight at 4°C with mouse monoclonal anti-β-catenin antibody (#sc-7963, Santa Cruz Biotechnology) and goat polyclonal anti-lamin B antibody (#sc-6216, Santa Cruz Biotechnology). Secondary antibodies were Alexa Fluor 647-conjugated anti-mouse IgG (#4410, Cell Signaling Technology), and FITC-conjugated anti-goat IgG (#sc-2024, Santa Cruz Biotechnology). The DNA dye Hoechst 33342 was used at 1 µg/ml to stain chromatin. Images were collected using a Nikon A1R confocal laser scanning microscopy system.