Alexa fluor 594 conjugate secondary antibody anti rabbit

For immunofluorescence, NP cells were seeded on slides in a six-well plate at a density of 4×10⁵ per well for 8 h. After 8 h, the NP cells were pretreated with or without DHE (2.5 μM) for 2 h and then stimulated with TNF-α (20 ng/ml) for 20 min. For dsDNA in the cytoplasm, the NP cells were treated with TNF-α (20 ng/ml) with or without DHE (2.5 μM) for 8 h. Para-formaldehyde (4%) was used to fix the NP cells for 15 min at RT, and 0.25% Triton X-100 was used to permeabilize the cells membrane for 5 min after washing three times with PBS. After blocking with 10% goat serum, the cells were incubated with a primary antibody for phospho-p65 (diluted 1:200, purchased from Cell Signaling Technology, anvers, MA, USA) or incubated with a primary antibody for dsDNA (diluted 1:200, purchased from Abcam, Cambridge, United Kingdom) overnight at 4°C. The next day, the cells were washed three times with PBS. Then, they were incubated with an Alexa Fluor 594 conjugate secondary antibody (anti-rabbit, 1:500; Cell Signaling Technology, anvers, MA, United States) for 1 h and subsequently incubated with DAPI (Sigma-Aldrich, St. Louis, MO, United States) for 3 min. Finally, digital fluorescence images were captured with a fluorescence microscope (Olympus, Inc., Tokyo, Japan).