Dylight 488 conjugated goat anti mouse igg

The staining of tissue sections and quantification of the staining were performed as we had previously described.³⁶ Cultured cells and tissue sections were fixed in paraformaldehyde (4%), followed by permeabilization with Triton X‐100 (0.1%) in PBS for 30 min, and blocked with goat serum (3%) for 2 h. Brain slices were incubated at 4°C overnight with the following primary antibodies: anti‐NeuN (1:200, mouse, 94403 S, Cell Signaling Technology, Danvers, MA, USA) and anti‐CXCR7 (1:200, rabbit, NBp2‐24,779, Novus Biologicals, Littleton, CO, USA). The slides were washed with PBS and incubated with DyLight 594‐conjugated goat anti‐rabbit IgG (1:500, E032420‐01, EarthOx, San Francisco, CA, USA) or DyLight 488‐conjugated goat anti‐mouse IgG (1:500, E032210‐01, EarthOx, San Francisco, CA, USA) in the dark for 2 h at room temperature. After staining with DAPI (P0131‐25 ml, Beyotime, Shanghai, China), all slides were read and imaged using a confocal microscope (LSM800, ZEISS, Germany).

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).