Immunol staining fix solution

RAW264.7 cells or peritoneal macrophages derived from C57BL/6 or TLR4^−/− mice were plated overnight on 20-mm glass-bottom cell culture dishes. Cells were treated with EspC (5 μg/mL) for 30 min, fixed and permeabilized in Immunol Staining Fix Solution (Biyuntian) for 20 min, and blocked with 3% bovine serum albumin in PBS. After washing with PBS, the treated RAW264.7 cells were incubated with anti-rabbit TLR2, anti-rabbit TLR4, and anti-mouse HIS antibodies for 2 h at 37°C in Immunol Staining Primary Antibody Dilution Buffer (Biyuntian). Alternatively, the treated peritoneal macrophages derived from C57BL/6 or TLR4^−/− mice were incubated with anti-rat TLR4 and anti-mouse HIS antibodies under the same conditions. After washing, the cells were restained with appropriate Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibodies for 1 h at 37°C. Cells were then stained with 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. The cells were analyzed by confocal microscopy using a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) with a 100 × oil immersion objective. Images were acquired and processed using LAS AF Lite software.

RAW264.7 cells were plated overnight on 20-mm glass-bottomed cell culture dishes. After treatment with EspC, the cells were incubated with the pre-warmed medium containing 250 nM of MitoSpy Red CMXRos (Biolegend) for 20 min. After the cells were washed with PBS, they were fixed and permeabilized in Immunol Staining Fix solution (Biyuntian) for 20 min and blocked with 3% bovine serum albumin (BSA) in PBS. The cells were washed with PBS and incubated with anti-cytochrome c and anti-Bax for 2 h at 37 °C in the Immunol staining primary antibody dilution buffer (Biyuntian). After the cells were washed with PBS, they were re-stained with the appropriate Alexa Fluor 488-conjugated secondary antibodies for 1 h at 37 °C and stained with 0.1 µg/mL DAPI for 10 min at 25 °C. The cells were analyzed using a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) (63X; NA, 1.4) oil immersion lens (HC PL APO CS2; zoom, 2.5X; speed, 400 Hz; line average and resolution, 4). Images were acquired and processed using the LAS AF Lite software.

DC2.4 cells were seeded in a 6-well plate at a density of 1 × 10⁵ cells/mL and then incubated in complete RPMI-1640 medium overnight. After pre-treatment with GSLS-NPs, G-Solution or empty NPs at a final concentration of 100 μg/mL for 4 h, the cells were incubated with the inactivated PEDV antigen for 24 h. Untreated cells served as the NC. After removing the supernatant, the cells were washed three times with PBS and fixed with Immunol Staining Fix Solution (P0098, Beyotime, Shanghai, China). A monoclonal antibody (1:100) against the PEDV nucleocapsid protein was then added and incubated for 1.5 h at 37 °C. DyLight 488-conjugated goat anti-mouse IgG (E032210-01, EARTHOX, San Francisco, CA, USA) (1:200) was used as the secondary antibody. LysoTracker Red (C1046, Beyotime, Shanghai, China) and DAPI (C1005, Beyotime, Shanghai, China) were used to stain the lysosomes and nuclei, respectively. The cells were observed under an inverse confocal laser scanning microscope (FV1000, Olympus, Tokyo, Japan). The images were acquired using Olympus confocal software (FV10-ASW 3.1). In addition, a monoclonal antibody against the PEDV nucleocapsid protein was labeled with the fluorescent dye APC by using an APC conjugation kit (ab201807, Abcam, Shanghai, China). The relative fluorescence intensity of intracellular PEDV was quantified on a FACSCanto^TM (BD Biosciences, San Diego, CA, USA).