Alexa fluor 594 conjugated goat anti mouse igg h l

GCO cells were cultured in M199 medium (HyClone, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL of penicillin, and 100 mg/mL of streptomycin under humidified conditions with 5% CO₂ at 28°C. Grass carp reovirus, which was isolated and identified as subtype I by our laboratory (50 (link)), was used in the study of viral infection. Rabbit polyclonal antibodies against the GCRV nonstructural protein NS80 (anti-NS80) and structural protein VP5 (anti-VP5) were prepared in our laboratory (51 (link)). Mouse anti-double-stranded RNA (J2 clone) antibody was purchased from Nordic-MUbio (Susteren, Netherlands). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L) was purchased from Cell Signaling Technology (Danvers, MA, USA). Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L) was purchased from Abcam (Cambridge, UK). The LD probes oil red O and Bodipy 493/503 were purchased from Beyotime (Shanghai, China) and GLPBIO (CA, USA). C75, TOFA, IBMX, palmitic acid (PA), atglistatin, and CAY10499 were purchased from GLPBIO (CA, USA). Triacsin C and isoproterenol were purchased from Abcam and MedChemExpress (USA).

Vero cells or porcine alveolar macrophages (PAMs) were plated in 24-well cell culture plates 24 h prior to infecting with the ASFV HLJ/18 strain at an MOI of 0.01. At 12 and 24 hpi, the infected cells were fixed using 4% paraformaldehyde for 30 min at RT, permeabilized using 0.3% Triton X-100 for 3 min at RT, and then blocked using 5% BSA for 2 h at RT. Then, the samples were washed 3 times using PBS and followed by incubating with Nb8-HRP supernatants and Alexa Fluor-594 conjugated goat anti-mouse IgG (H&L) (Abcam). Nuclei were stained with DAPI, and the images were obtained using an inverted fluorescence microscope (Olympus Corporation). The ASFV-infected cell samples were inactivated and kindly provided by Harbin Veterinary Research Institute and were stored at −20°C during shipping.

Mouse monoclonal antibody (mAb) directly against His and rabbit polyclonal antibodies (pAb) directly against α-tubulin were purchased from Proteintech. Mouse mAb 6D10, which specifically reacts with the PRRSV N protein (1 μg/mL), has been described in our previous work [39 (link)]. HRP-conjugated goat anti-mouse and -rabbit IgG antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc (West Grove, PA, USA). Alexa Fluor 594 conjugated goat anti-mouse IgG H&L and Alexa Fluor 488-conjugated goat anti-rabbit IgG H&L were both purchased from Abcam (Cambridge, UK).

To observe the expression of HN protein, 1 μg of original HNs or mutant HNs was transfected into BHK-21 cells by using Lipofectamine 2000 Transfectant Reagent (Invitrogen, CA, USA). At 24 h post-transfection, each well was fixed with PBS containing 4% paraformaldehyde and blocked with PBS containing 1% bovine serum albumin (BSA). Then, the cells were incubated with an HN monoclonal antibody made by our laboratory at an optimized dilution of 1:1000 and incubated with Alexa Fluor^® 594-conjugated goat anti-mouse IgG (H + L) at a 1:500 dilution (Abcam, Shanghai, China) as the secondary antibody. Photographs of the cells were recorded under a fluorescence microscope (IX73; OLYMPUS, Tokyo, Japan). Empty cells were used as mock controls, and empty pCAGGS was used as the negative control. For rescued viruses, BHK-21 cells were incubated with viruses at an MOI of 0.1 for 12 h or 24 h. An IIFA at the virus level was performed with the methods described above.