N sim e

Cells were cultured on coverslips, fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X‐100 (PBS) for 5 min, blocked with 1% (w/v) BSA (PBS) for 1 h, and then incubated with primary antibodies overnight. After washing, cells were incubated with fluorescence-labeled secondary antibody for 1 h at room temperature. The coverslips with cells were sealed with Fluor shield mounting medium with DAPI and imaged using a Super-Resolution Microscope (NIKON, N-SIM E). The number and size of p62 bodies were analyzed with the Image J software.
The primary antibodies were used: Smurf1 (Santa, sc100616; Abcam, ab57573), Keap1 (Proteintech, 10503-2-AP), Keap1 (Abcam, ab118285), p62 (Enzo, BML-PW9860), p62 (MBL, PM045), p62 (MBL, M162-3), LC3B (Sigma, L7543), LC3B (Novus, NB100-2220), NBR1 (Proteintech, 160004-1-AP), HA (MBL, M180-3), Nrf2 (Proteintech, 16396-1-AP), Flag (Sigma, F1804).
The secondary antibodies were used: Alexa Fluor^® 555 goat anti-mouse IgG (Life Technologies, A21425). Alexa Fluor^® 488 goat anti-rabbit IgG (Life technologies, A11008), Alexa Fluor^® 555 goat anti-rabbit IgG (Life technologies, A21428), Alexa Fluor^® 488 goat anti-mouse IgG (Life technologies, A11001), Alexa Fluor^® 647 goat anti-mouse IgG (Abcam, ab150115), Alexa Fluor^® 647 donkey anti-goat IgG (Abcam, ab150131), Alexa Fluor^® 405 goat anti-rabbit IgG (Abcam, ab175652).

After the imaging of in vivo leakiness, the brains were fixed in 4% paraformaldehyde solution and removed after 48 h. Sucrose (30%) was added overnight for dehydration after brain fixation. They were then embedded with optimal cutting temperature (OCT) compound and sliced in 5 μm-thick transverse sections by a freezing microtome. Then, slices were incubated with 10% skimmed milk for 2 h, then incubated with anti-Aβ antibody (1:250 dilution) overnight. Alexa Fluor 488 or 647-conjugated secondary antibody (1:250 dilution) was used for fluorescence detection. DAPI was used for the visualization of nuclei and EBD was also observed at 555 nm with a super-resolution laser confocal microscope (Nikon, N-SIME).

HUVECs were seeded in confocal dishes at a density of 8 × 10⁴ cells/well and cultivated for 24 h. Cells were treated with PS nanoplastic (0.05 or 0.5 mg/mL) for 6 h. Then, cells were stained with DAPI (200 μL) for 10 min. After washing with PBS three times, morphological features of the cells were captured using a confocal fluorescence microscope (Nikon, N-SIM E).

To obtain confocal laser scanning micrographs, a Leica TCS‐SP5 system was used. Micrographs were acquired using a 40× 1.2 NA oil immersion objective with a voxel size of 189 × 189 × 500 nm (xyz). For whole‐slide near‐infrared imaging, an Olympus FV3000 system with a 20×, 0.8 NA air objective equipped with a 405, 488, 561 and 640 nm laser lines and an external NIR‐unit with a 730 nm laser line was used. The whole section was imaged as tiled stacks over 2 µm with a voxel size of 222 × 222 × 500 nm. Tile data were stitched within the Olympus FV3000 CellSense software. For super‐resolution 3D‐structured illumination microscopy, a Zeiss Elyra PS.1 system (Carl Zeiss Microsystems) or a Nikon N‐SIM‐E was used as described before.³ Whole‐slide images of PAS‐stained sections were acquired on a Leica SCN400 slidescanner. SCN files were imported and processed with QuPath (v0.3.0).

RBCs were labeled with CellMask deep red stain (1:3000) for 15 min at rt and washed with PBS by centrifugation at 800× g for 10 min. Pelleted RBCs 4 × 10⁵ cells/well were dissolved in FluoroBrite DMEM and added to a final volume of 300 µL/well in 8-well chamber slides. Stx1B:Alexa-488 (10 ng/mL, this concentration was optimized to achieve a low fluorescent background and a detectible Stx1B signal) was added to the cell medium during live cell imaging. Cells were imaged in a Nikon Ti-E microscope (×100 oil objective) equipped with N-SIM E (Nikon Instruments Inc., Tokyo, Japan) and a Hamamatsu Flash 4 camera (Hamamatsu photonics, Shizuoka, Japan) using NIS elements AR software V.5.11.01 (Nikon Instruments Inc.).