Confocal microscopy

In the cell uptake study, G, MG, SWCNTs-G, and SWCNTs-MG were incubated with 6 × 10⁷ macrophage for 24 h. Macrophages were obtained by centrifugation at 1500 rpm for 2 min. Furthermore, treated macrophages were immunostained with the tissue resident macrophage marker F4/80 primary antibody (1:250, Abcam, Cambridge, England), Cy3-labeled secondary antibody (1:1000, Beyotime. China), and DAPI (Beyotime. China). The cell uptake of fluorescently labeled nanovaccine was analyzed by BD FACSAria flow cytometry (BD, USA) and confocal microscopy (Leica, Germany).
For the detection of nanovaccine in vaccinated fish, carps (80 tail in total) were randomly divided into 4 groups (20 tail/group): G, MG, SWCNTs-G, and SWCNTs-MG group. Each group was immersed with G-FITC, MG-FITC, SWCNTs-G-FITC, and SWCNTs-MG-FITC at 30 mg/L for 6 h, respectively. After vaccination, fishes were transferred to clean water. Tissues including muscle, gill, intestine, kidney, spleen, and liver were isolated from vaccinated fish. Then tissues sections were made and then observed in confocal microscopy (Leica, Germany). Image J software was used to quantify the intensity of fluorescence in each group.

NO production was measured using the commercial kit (Jiancheng Bioengineering Institute) as we described.²⁵In situ NO production was detected using DAF‐FM diacetate (Molecular Probes). Isolated aortas were embedded and froze in OCT medium. The cryosections were incubated in PBS with DAF‐FM diacetate (10 μM) for 20 minutes at room temperature. Images were recorded using a confocal microscopy (Leica). Intracellular NO level was assessed using DAF‐FM diacetate (Molecular Probes).²⁹ Briefly, the cells with different treatments were rinsed with PBS in the glass chamber. Then, the cells were incubated with DAF‐FM diacetate (10 μM) for 10 minutes at 37°C. Finally, the fluorescence pictures were recorded using a confocal microscopy (Leica). The staining density was quantified using Image‐J software.

For immunohistological staining, muscle sections were fixed by 3% H₂O₂, and blocked for 20 min at room temperature by goat serum (HyClone). Anti-Fbxo32 (ECM Bioscience, 1:500) antibody was used as primary antibody. Secondary antibodies (Jackson Laboratory) were diluted 1:2000 in blocking buffer and incubated for 1 h at room temperature. Mixture of substrate A (Dako) and B (Dako) were diluted in blocking buffer and incubated for 30 min at room temperature, and developed by DAB+ Chromogen (Dako). Images were acquired by confocal microscopy (Leica). For immunofluorescent staining, cells or sections were fixed with 4% parafamaldehyde and permeabilized with cold methanol, anti-MYH3 (Developmental Studies Hybridoma Bank, clone F1.652, 1:1000), anti-MYHC (Merk Millipore, clone 05-716, 1:1000) and anti-lammnin (Abcam, clone B00648, 1:1000) antibodies were used as primary antibody, respectively. The cells and sections were next stained with Alexa 488-, 561- or 647-labeled anti-mouse or -rabbit antibodies (Invitrogen). All images were acquired by confocal microscopy (Leica).