P62 antibody

RAW264.7 cells (5 × 10⁵ cells) were seeded and incubated with LPS or LPS in the presence of LDL. The cells were treated with DS-Ce6 (equiv. 5 μM Ce6) for 1 h, irradiated using a 670 nm laser (50 mW, irradiated area 3.8 cm²) for 10 s, and further incubated for 15 min, 30 min, and 1 h. Cells without laser irradiation were used as a control. The cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 10 min at room temperature, blocked with 1% BSA for 30 min, and incubated with LC3 antibody (1:200; Novus Biologicals) for 1 h at room temperature, followed by incubation with Alexa Fluor 594 AffiniPure goat anti-rabbit IgG (1:100) in the dark. After washing three times with PBS for 5 min each in the dark, the cells were incubated with p62 antibody (1:200; Abcam) for 1 h at room temperature and then with Alexa Fluor 488 AffiniPure goat anti-rabbit IgG (1:100) in the dark. After washing with PBS, the cells were mounted using a fluorescence mounting medium with DAPI. Fluorescence was observed using a model LSM 780 Meta NLO confocal microscope (Carl Zeiss). The LC3 puncta per cell of at least 20 cells in each experimental arm was counted by a blinded observer (n = 5).

Brain microvascular endothelial cells that had undergone the different treatments for 48 h were seeded into 6-well plates, washed with phosphate buffer solution (PBS), and then fixed with 4% paraformaldehyde (PFA) at 4°C overnight. Next, the cells were washed twice with PBS (3 min per wash), blocked with 10% goat serum for 15 min, and incubated with LC3B antibodies (1:200, Abcam, Cambridge, MA, United States) or P62 antibodies (1:50, Abcam, Cambridge, MA, United States) at 4°C for 1 h. After being washed three times with PBS (3 min per wash), the cells were incubated with Alexa Fluor 488 (for P62) or Alex Fluor 594 (for LC3B) conjugated secondary antibodies (1:1000, Abcam, Cambridge, MA, United States) at room temperature for 1 h. Next, the secondary antibodies were removed, and the cells were washed three times with PBS (5 min per wash). Finally, the cells were mounted with anti-fluorescence quench sealing liquid, and analyzed under an inverted florescence microscope (Bx51. Olympus Corporation, Shinjuku, Japan).

As previously described [14 (link)], cells from each group were collected and placed in a 1.5 mL EP tube, followed by addition of appropriate amount of lysate for completely lysis and ultrasonic testing in the ice bath sing ultrasonic processor; after centrifugation at 2000 rpm for 15 min at 4 °C, the supernatant was extracted and stored at − 20 °C for further use. The oncentration of proteins extracted above was quantified sing BCA Protein Quantification Kit; 5 × loading buffer as added to the sample, followed by boiling at 100 °C or 5 min to denature the proteins. After processing by DS-PAGE, the proteins were transferred to the NC embrane using protein transfer device and then locked by 5% skimmed milk powder for 1 h. After addition of p62 antibodies (Abcam UK) and incubation vernight at 4 °C, the proteins were washed by BST three times before 1 h of fluorescent secondary ntibody incubation and another three times of TBST ashing; Western Blot imaging software Odyssey nalyzer was used to detect and produce images.