Rabbit monoclonal antibody against nf κb p65

HDPCs were plated onto coverslips at a density of 2 × 10⁴ cells/cm². After 24 hrs, the cells were treated with TNF‐α for respective timescales. Briefly, the cells were fixed in 4% paraformaldehyde for 10 min. at room temperature, washed in PBS, permeabilized with 0.1% triton for 5 min. and blocked with 4% BSA for 30 min. at 37°C. Subsequently, the slides were incubated with primary rabbit polyclonal antibody against Panx3 (1:25; Invitrogen, Carlsbad, CA, USA) and rabbit monoclonal antibody against NF‐κB‐p65 (1:100; Cell Signaling Technology, Danvers, MA, USA) at 4°C in a moist chamber overnight. After three washes, the slides were incubated with DyLight 549‐conjugated goat anti‐rabbit antibody (1:200; Abbkine, Redlands, CA, USA) for 60 min. at 37°C. After three washes, nuclei were then stained with 4, 6‐diamidino‐2‐phenylindole (DAPI). For control, the primary antibody was omitted.

At 24 hours after reperfusion, protein extraction reagents (Beyotime Biotech. Co., China) were used for extraction of nuclear and cytosolic proteins of the tissue samples according to the manufacturer's instructions. Whole protein weighing 50 μg was used for measurement of the content of TLR4. Nuclear protein weighing 15 μg was used for evaluation of the content of the NF-κB p65 subunit. Protein samples were separated on 10% SDS polyacrylamide gels and transferred to nitrocellulose membranes. The nonspecific binding sites were blocked with 5% nonfat dry milk Tris-buffer. The membranes were subsequently incubated overnight at 4°C with a rabbit polyclonal antibody against TLR4 (1 : 250; Novus Biologicals) or a rabbit monoclonal antibody against NF-κB p65 (1 : 1000; Cell Signaling Technology), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1 : 2,0000, Jackson, American). The protein bands were visualized by a chemiluminescence detection system and exposed on an X-ray film. The expressions of TLR4 and NF-κB p65 were normalized to β-actin to correct the variations of different samples. The optical densities of protein bands were measured by an image analysis software (Image Pro Plus 6.0).

GECs were grown on 18 mm glass coverslips and infected with C. pneumoniae at a MOI of 5. After three days, the cells were washed, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS for 40 minutes. Following one wash with PBST (0.05% Tween-20 in PBS) and two washes with PBS, the cells were blocked with 2% BSA in PBS for 30 minutes before hybridization overnight at 4°C with a rabbit monoclonal antibody against NF-κB/p65 (Cell Signaling, catalog #8242). Cells were again washed once with PBST followed by two PBS washes before incubation with Alexa Fluor 546-conjugated goat anti-rabbit IgG (Invitrogen) for 40 minutes. Cell nuclei were counterstained with 2 µg/ml Hoechst 33342 (Sigma-Aldrich). Fluorescence images were captured using a DMI4000B confocal microscope (Leica) and analyzed using LAS-AF software (Leica).