Alexa fluor 488 labeled goat anti mouse igg

The A/Swine/Shanghai/3/2014(SH/2014) H1N1 strain was isolated from a clinical pig with the symptoms of swine influenza. Madin-Darby canine kidney (MDCK) cells and the human epithelial kidney cell line (293 T) were used to rescue reassortant viruses from plasmids and were then cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) plus antibiotics. Mouse monoclonal antibodies against IRF3 and phospho-IRF3 (p-IRF3) were purchased from Cell Signaling Technology (Danvers, MA, USA), and a mouse monoclonal anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody was purchased from Jackson ImmunoResearch (West Grove, PA, USA). Alexa Fluor 488-labeled goat anti-mouse IgG was purchased from Beyotime Biotechnology (Nantong, China). A mouse polyclonal antibody against the NS1 protein was kindly provided by Professor Ying Fang at Kansas State University, USA.

The H1N1 SIV A/swine/Shanghai/3/2014(SH/2014) strain was isolated during routine surveillance and sequenced by our laboratory. Madin-Darby canine kidney (MDCK) cells and the human epithelial kidney cell line (293 T) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA, USA) at 37 °C and 5% CO₂. Rabbit polyclonal antibodies against RIG-I and horseradish peroxidase (HRP) conjugated goat anti-rabbit or anti-mouse secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). A mouse monoclonal antibody against β-actin was purchased from Sigma (St Louis, MI, USA). Alexa Fluor 488-labeled goat anti-mouse IgG was purchased from Beyotime Biotechnology (Nantong, China). A mouse polyclonal antibody against the NS1 protein was generously provided by Professor Ying Fang at Kansas State University, USA.

4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) (sc-202511), 5-Tetradecyloxy-2-furonic acid (TOFA) (sc-200653), and etomoxir (sc-208284) were purchased from Santa Cruz Biotechnology. Trimetazidine (TMZ) (S61054) was purchased from Shyuanye Biotechnology. High glucose dulbecco's modified eagle medium (DMEM) (11995040), low glucose DMEM (11885084), no glucose DMEM (11885084), high glucose DMEM with no glutamine (11960069), RPMI 1640 medium (21875091), glucose free RPMI 1640 medium (11879020), and glutamine free RPMI 1640 medium (21870076) were obtained from Gibco. The following primary antibodies were used in the study: rabbit monoclonal anti-MDA5 (Sigma-Aldrich, SAB2101127), rabbit polyclonal anti-RIG-I (Cell Signaling Technology, 3743), rabbit polyclonal anti-NF-κB (Beyotime, AF0246), rabbit monoclonal anti-Phospho-IRF3 (Beyotime, AF1594), and mouse monoclonal anti-GAPDH (Beyotime, AG019) and mouse monoclonal anti-CSFV E2 (WH303) (JBT, 9011). Mouse polyclonal anti-CSFV Npro was kindly provided by Dr. Xinglong Yu (Veterinary Department, Hunan Agricultural University, China). Te secondary antibodies were used in the study: HRP-conjugated goat anti-mouse IgG (Beyotime, A0192), HRP-conjugated goat anti-rabbit IgG (Beyotime, A0208) and Alexa Fluor 488-labeled Goat Anti-Mouse IgG (Beyotime, A0428).

Under an anatomical microscope, the mucosa and submucosa of colonic tissues were removed as a layer from the colonic tissues fixed at 4% paraformaldehyde to prepare full-thickness tissue specimens of colonic musculus, and the full-thickness tissue of the colonic musculus was used for immunofluorescence staining. The specimens were antigen repaired and sealed by bovine serum albumin for 1 h. Then the primary anti-AHR (1:200, cat. No. sc-133088) and anti-CYP1A1 (1:200, cat. No. sc-393979) antibodies (Santa Cruz Biotechnology, USA) were incubated with anti-PGP9.5 antibody (1:200, cat. No. ab10404, Abcam, UK) at 4 °C overnight, and the fluorescent secondary antibodies Alexa Fluor 647-labeled Goat Anti-Rabbit IgG(H + L) (1:500; cat. No. A0468; Beyotime Biotechnology) and Alexa Fluor 488-labeled Goat Anti-Mouse IgG (H + L) (1:500; cat. No A0428; Beyotime Biotechnology) were used to incubating at room temperature and away from light for 1 h. Finally, the 4′,6-diamidino-2-phenylindole (DAPI, Sigma, USA) was used for re-staining the nucleus for 5 min, and images were scanned and obtained under OlyVIA (OLYMPUS, Japan). The Integrated Density (IntDen) and Area of each image were determined by image-J analysis system (National Institutes of Health, USA), and the mean fluorescence intensity of each image was calculated.

MARC-145 cells were infected with PRRSV at a multiplicity of infection (MOI) of 0.1 or transfected with an infectious cDNA clone. After 24 h, the cells were fixed with 4% paraformaldehyde for 15 min, then permeabilized with methanol for 10 min. Next, the cells were washed three times with phosphate-buffered saline (PBS) and blocked using 5% bovine serum albumin (BSA) at 37 °C for 1 h. Following three washes with PBS, the cells were incubated with monoclonal antibody specific to PRRSV-N protein at 37 °C for 1 h. Afterwards, the cells were stained with Alexa Fluor 488-labeled Goat Anti-Mouse IgG (Beyotime) at 37 °C for 45 min. The nuclei were stained with 0.01% 4′,6-diamidino-2-phenylindole for 15 min. After three washes with PBS, fluorescent images were visualized and collected using a fluorescence microscope (Nikon, Tokyo, Japan).