S 1000 20

After 30 min pretreatment with hispidulin (5 µM), HUVECs were exposed to P. gingivalis LPS (5 µg/mL) for 1 h and then fixed in 4% paraformaldehyde for 10 min. Subsequently, the cells were blocked using a 0.5% Triton X-100/PBS and 5% normal goat serum (#S-1000-20, Vector Labs, Burlingame, CA, USA) and reacted with the primary antibody against NF-κB p65 (diluted 1:100, #SC-8008, Santa Cruz Biotechnology) for 1 h. After incubation with the primary antibody, the cells were rinsed three times for 10 min with PBS and incubated with the Alexa^® 488-conjugated secondary antibody (diluted 1:200, #A11001, Invitrogen) for 1 h in the dark. Coverslips were mounted with DAPI-containing Vectastain (#H-1200, Vector Laboratories). Cell analysis was conducted using a confocal microscope (LSM900; Zeiss, Oberkochen, Germany).

For ICC, 1 × 10⁴ cells from each of the human pancreatic cancer and normal cell lines were seeded on cover glasses overnight and fixed with 2% formaldehyde. Antigen retrieval was carried out in 10 mM sodium citrate buffer. Samples were then blocked with normal goat serum blocking solution (S-1000-20, Vector Laboratories, Burlingame, CA) and treated with primary antibodies (Supplemental Table S2). Biotinylated or Alexa Fluor™-conjugated secondary antibodies were applied according to primary antibody host species (Supplemental Table S2). Sections for IHC were treated with horseradish peroxidase streptavidin (SA-5004-1, Vector Laboratories) and a DAB substrate kit (34065, ThermoScientific, Waltham, MA) and counter-stained with hematoxylin.

Cells were fixed in 4% paraformaldehyde at 37 °C for 20 min, permeabilized with 0.2% Triton X-100 in 1X PBS at 37 °C for 30 min, and incubated with blocking buffer consisting of 5% normal goat serum (Vector Labs, S-1000-20). Cells were stained with antibodies against Cytochrome c (1:100, BD Pharmingen #556432) or Tom20 (1:100, Santa Cruz, sc-11415) overnight at 4 °C. Cells were then rinsed with 1X PBS and incubated with an Alexa Fluor 594 secondary antibody (1:200, Invitrogen, A11037) for 1 h at 37 °C. Lastly, cells were stained with Hoechst 33342 (1:1000, Invitrogen, H3570) to label nuclei.
Fluorescence images were captured using a Nikon Eclipse Ti2-E with a motorized XYZ-stage fitted with a Plan-Apochromat lambda 60X NA 1.40 oil immersion objective. Z stacks were separated by 0.3 mm and acquired with a DS-Qi2 camera (Nikon Instruments, Melville, NY, USA) illuminated by a solid-state white light excitation source (Lumencor, Beaverton, OR, USA). Images were processed by deconvolution and compressed into extended depth-of-focus (EDF) images by NIS-Elements AR GA3 software. To evaluate mitochondrial clearance, 200 cells were counted per condition in each experiment. To assess GFP-LC3 and mitochondrial colocalization, the number of GFP-LC3 puncta overlapping with mitochondria was manually scored. A minimum of 20 cells were scored per condition in each experiment.

To conduct immunofluorescence staining, brain sections were rinsed in 0.2% PBST (PBS + 0.2% Triton-X 100) and incubated in blocking solution [10% normal goat serum (Vector Laboratories, S-1000-20, Burlingame, CA) in PBST (PBS + 0.2% Triton-X 100)] for 2 h at room temperature. Next, the tissue sections were incubated with primary antibodies at 4°C for 24–72 h, washed with PBST, and incubated with fluorescent goat anti-rabbit, anti-mouse or anti-chicken secondary antibodies conjugated with Alexa Fluor 488 or 555 for 2 h at room temperature. Finally, the brain sections were washed with PBST, PBST/DAPI, and PBS and mounted on glass slides with a mounting solution containing DAPI (Vector Laboratories, H-1200-10, Burlingame, CA). Images of the immunostained tissue were obtained by fluorescence microscopy (DMi8, Leica Microsystems) and quantified using ImageJ software (US National Institutes of Health, Bethesda, MD, USA). The primary and secondary antibodies are listed in Table 2.