F4 80 primary antibody

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.

F4/80-postive cells in iWAT and eWAT of HFD adipo-Gi KO mice and control littermates were identified via immunostaining, using the anti-rat HRP-DAB Staining Kit (R&D System). Briefly, slides/sections were blocked with peroxide blocking reagent for 5 min, followed by a 15 min incubation with Serum-Blocking Reagent G. Subsequently, slides were incubated with Avidin Blocking Reagent for 15 min, rinsed with PBS, treated with Biotin Blocking Reagent for 15 min, and then incubated with the F4/80 primary antibody (1:200, Abcam) at 4 °C overnight. On the next day, slides were rinsed with PBS, treated with biotinylated secondary antibody for 60 min, and then incubated with High Sensitivity Streptavidin-HRP for 30 min. Finally, the slides were developed using DAB and counterstained with hematoxylin. Bright-field images of stained tissue sections were taken with a Keyence Microscope BZ-9000.