Fitc conjugated goat anti mouse igg

Indirect immunofluorescence assays (IFAs) were performed in both extracellular and intracellular N. caninum tachyzoites. Free N. caninum tachyzoites (Nc-1) were washed three times with PBS, added to poly-L-lysine (0.1 mg/ml, Sigma Aldrich) precoated cover glass slides, dried at RT and fixed in 4% paraformaldehyde for 10 min. The cover slides were then washed three times with PBS, permeabilized with 0.25% Triton X-100 in PBS for 10 min, washed and blocked in 3% BSA/PBS for 2 h at RT. After blocking, the samples were incubated with a 1:100 dilution of the 14-3-3 antibody overnight at 4°C, then washed and incubated with the secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ig-G (Proteintech, Wuhan, China) for 1 h at RT. The coverslips were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Thermo Scientific, Waltham, MA, United States) for 10 min before analysis on an Olympus FV1000 Laser Scanning Confocal microscope (Japan).
Neospora caninum tachyzoite-infected VERO cells were stained with the 14-3-3 antibody as follows. The intracellular tachyzoites were washed with cold PBS three times and fixed on glass coverslips with 4% formaldehyde for 20 min at 4°C. The infected cells were permeabilized with 0.25% Triton X-100 in PBS for 8 min, washed and blocked in 3% BSA/PBS for 2 h at RT. All procedures were the same as those described above.

For immunohistochemical (IHC) staining, tissues were embedded in paraffin after fixed in 4% paraformaldehyde and sectioned at 4 μM. After antigen retrieval at high temperature, sections were incubated with sheep serum albumin for blocking antigen. Sections were then incubated with primary antibodies (anti-ADH1B, anti-ALDOB, anti-ADH1A, anti-FBP1, and anti-ADH6; ABclonal Technology) for overnight. Secondary antibody was applied for 30 min. After added the diaminobenzidine solution, sections were stained with hematoxylin. The images were visualized by XSP-C204 biomicroscope.
Additionally, sections were stained with anti-mouse CD14 monoclonal antibody (Proteintech, Wuhan, China) for 15 h, then incubated with FITC-conjugated goat anti-mouse IgG (Proteintech) for 1 h. After washing with solution for 4 times, sections were incubated with sheep serum albumin for blocking antigen. Then sections were stained with anti-rat FOXP3 polyclonal antibody (Absin, Shanghai, China) for 15 h, then incubated with Cy3-conjugated goat anti-rat IgG (Proteintech) for 1 h. Nuclei were counterstained with DAPI. The images were visualized by XSP-C204 biomicroscope.