4 6 diamidino 2 phenylindole (dapi)

The following antibodies and reagents were used: pα-Syn (Ser129, Biolegend, 825701), pα-Syn (Ser129, Cell Signaling Technology, 23706s), MAP2 (Thermo Fisher Scientific, SF254293), COX IV (Abcam, ab16056), ATG5 (Proteintech, 10181-2-AP), Beclin1 (Proteintech, 11306-1-AP), LC3 (Cell Signaling Technology, 12741), Bcl2 (Cell Signaling Technology, 3498S), Bax (Proteintech, 50599-2-Ig), GAPDH (Proteintech, 60004-1-Ig), TH (Sigma-Aldrich, AB152), Ubiquitin (Santa Cruz Biotechnology, sc-8017), Iba-1 (Wako, 019-19741), Alexa Fluor 594-conjugated goat anti-mouse IgG (Invitrogen, A-11005), Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, A-11012), DAPI (Biofroxx, EZ3412B205), HRP-conjugated anti-mouse IgG (BIO-RAD, 170-6516), HRP-conjugated anti-rabbit IgG (BIO-RAD, 170-6515), Complex I Enzyme Activity Microplate Assay Kit (Abcam, ab109721), and Reactive Oxygen Species Assay Kit (Nanjing Jiancheng Bioengineering Institute, E004-1-1).

Colon sections were incubated with anti‐LC3II (1:100, #2775, CST) and anti‐GRP78 (1:100, 66574‐1‐lg, Protein Tech) antibodies. Secondary antibodies (FITC‐conjugated AffiniPure goat anti‐mouse IgG, BA1101, TRITC‐conjugated AffiniPure goat anti‐rabbit IgG, BA1090, Boster, USA) were used in this study.
Cells were incubated with anti‐LC3II (1:500). Nuclei were stained with DAPI (1155MG010, Biofroxx, Germany). Images were obtained using a LEICA SP8 confocal microscope (LEICA, Germany). And the fluorescence intensity was quantified using ImageJ software (Fiji, ImageJ).

BMSCs treated with various concentrations of C₂S NPs for 3, 6, and 12 h were coated overnight with 4% paraformaldehyde at 4 °C, permeabilized with 0.01% Triton X-100 for 20 min at room temperature, and blocked with 5% BSA for 30 min at 37 °C. After incubation with specific primary antibody LC3 (1 µg/ml Abcam, UK) overnight at 4 °C and treatment with fluorescent secondary antibodies (1:50; Cell Signaling Technology (USA)), all the specimens were rinsed and stored at room temperature for 1 h. The nucleus was counterstained with DAPI (Biofroxx, Germany) for 5 min at room temperature and then washed three times with PBS. The dyed cells were examined under a confocal microscope (Leica SP8, Germany). ImageJ software (NIH) was utilized to analyze cell images, where the width, perimeter, length, and area of the cells were determined, and the roundness (4π*Area/[perimeter]2) and aspect ratio (length/width) was calculated. Each sample was randomly selected under 3 fields of view analyzing 3 samples per group.

The physiological effects of FCLAPs on bacterial cells were evaluated using visualized fluorescent imaging. E. coli O157:H7 was cultured in MHIIB until the stationary phase. Cells were then incubated with FCLAPs (4 × MIC) for 1 h. Following AMP treatment, cells were stained with 20 µg mL⁻¹ FM4‐64 (Molecular Probes) and 10 µg mL⁻¹ DAPI (Biofroxx) for 10 min. Each stained culture was then centrifuged and resuspended in 1/10 volume of the original cultures. Five microliters of the concentrated cells were spotted onto a 1.5% agarose‐coated glass slide for microscopy. Images were collected using a ZEISS LSM 880 super‐resolution confocal microscope using a 63 × oil‐immersion objective lens.

Frozen histological sections of goat lip (health and ORFV-infected) or thymus tissues were prepared. First, slides were fixed with antigen retrieval bufferson for 10 min, and then blocked with 5% donkey serum for 2 h. Next, slides were incubated overnight at 4 ℃ with CD4 or CD25 fluorescent antibody. Finally, using DAPI (BioFROXX, Guangzhou, China) to stain the nuclei, and images were captured using confocal laser scanning microscopy (Leica TCS SP8, Germany). It should be noted that between each two steps of this experiment, the slides were washed with PBS seven times for 10 min each time.

4T1 cells were seeded in six-well plates (3 × 10⁵ cells/well). After 24 h, DiD+FAM-CpG-S or DiD+FAM-CpG-N/A (both with a DiD concentration of 1 μg/mL) was added and cells were incubated for 2 or 4 h, respectively. Afterward, the cells were digested, collected, and analyzed by flow cytometry (Beckman Coulter).
4T1 cells were seeded in six-well plates with a coverslip in each well and incubated with DiD+FAM-CpG-S or DiD+FAM-CpG-N/A as above. At 2 or 4 h post-treatment, cells were fixed with 4% polyformaldehyde, washed twice with phosphate-buffered saline (PBS), and stained with DAPI (0.5 μg/mL; Biofroxx, Einhausen, Germany) for 5 min. The fluorescence intensity was observed with a Nikon A1R+ confocal microscope (Tokyo Metropolis, Japan).