Fitc conjugated goat anti mouse igg

FHM cells were cultured in DMEM medium supplemented with 10% fetal bovine serum until reaching 80%–90% confluence. According to the manufacturer’ instructions, transfection agent of Fugene 6 (Promega, Shanghai, China) and plasmids were transfected into FHM cells at a ratio of 2:1 (v/v), before 4 μg of plasmid DNA was added to the medium in a 6-well plate. After 6 h of incubation, the medium was removed, and the cells were cultured in fresh medium at 28 °C with 5% CO₂. 36 h after transfection, cells were subsequently fixed with 4% (w/v) paraformaldehyde for 10 min, permeabilized with 0.1% (v/v) Triton X-100 for 10 min, and blocked the nonspecific binding with 3% (v/v) BSA at 37 °C for 1 h. The slides were washed three times with PBS and then incubated with mouse anti-HA antibodies (1:3000, ABclonal) and FITC-conjugated goat anti-mouse IgG (1:200, ABclonal) at 37 °C for 1 h, successively. The nuclei of all cells were stained with 1 mg/mL Hoechst 33,342 at room temperature for 10 min and photographed on UltraVIEW VoX 3D Live Cell Imaging System (PerkinElmer, Waltham, MA, USA). Cells transfected with pcDNA3.1 were used as negative control.

IFA tests on ASFV-infected cells were conducted on porcine alveolar macrophage (PAM) cells infected with ASFV (The virus was isolated and produced by PAM cells, and the virus TCID₅₀ was measured by the Reed–Muench method. The virus was stored at −80°C. The virus was isolated and stored in the Animal Biosafety Level 3 Laboratory of Huazhong Agricultural University.). PAM cells were collected from 20 to 30-day-old pigs, and the cells were plated on 96-well plates in 10% FBS (Gibco, Thermo Scientific, USA) RPMI 1640 medium (Gibco, Thermo Scientific, Waltham, MA, USA) at 37°C with 5% CO₂ and infected with ASFV at an MOI of 0.1. At 36 hpi, cell monolayers were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. The above operations are carried out in the Animal Biosafety Level 3 Laboratory of Huazhong Agricultural University. Cells were incubated with anti-p30 mAb followed by incubation with FITC conjugated goat anti-mouse IgG (ABclonal Technology Co., Ltd., Wuhan, China). Nuclei were stained with DAPI, and the plates were examined using the fluorescence microscope (EVOS FL Auto, Thermo Fisher Scientific, USA).

Leukocytes, erythrocytes, and CIK cells after GCRV infection for 0 h, 12 h, 24 h, 36 h, and 48 h were collected into a centrifuge tube and washed in staining buffer (phosphate buffered saline (PBS) + 3% BSA). Before intracellular staining, the cells were fixed in paraformaldehyde for 10 min, and permeabilized by incubation in Triton X-100 for 10 min. Cells were stained with Anti-VP4 (1:500) for 2 h and secondary FITC-conjugated goat anti-mouse IgG (1:50, ABclonal, Wuhan, China) for 1 h. All washing solutions and dilutions are diluted with staining buffer. The cells were read on a Cytoflex S Flow Cytometer (Beckman Coulter, California, America), counting 10,000 cells per sample.

The leukocytes, erythrocytes, and CIK cells after GCRV infection for 48 h were fixed with 4% (v/v) paraformaldehyde for 5 min, permeabilized with 0.1% (v/v) Triton X-100 for 10 min. Subsequently, they were blocked with the non-specific binding with 2% (v/v) BSA at 37 °C for 2 h. The slides were washed three times with PBST and then incubated with mouse anti-vp4 antibodies (1:1000) and FITC-conjugated goat anti-mouse IgG (1:200, ABclonal, Wuhan, China) at 37 °C for 2 h and 45 min. The nuclei of all cells were stained with 1 μg/mL Hoechst 33,342 at room temperature for 10 min. Afterward, the stained cells were rinsed with PBS. Images were taken with an UltraVIEW VoX 3D Live Cell Imaging System (PerkinElmer, Waltham, MA, USA).