Anti-NF-kB p65 antibody (ab16502)

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The deparaffinized sections were submerged in methanol containing 0.3% hydrogen peroxide for 15 min at room temperature to inhibit endogenous peroxidase activity. Antigen retrieval was done prior to incubation with a rabbit anti-NF-κB p50 polyclonal antibody (1:100 dilution; ab7971, Abcam), a rabbit anti-NF-κB p65 polyclonal antibody (1:500 dilution; ab7970, Abcam), a rabbit anti-matrix metalloproteinase (MMP)-3 polyclonal antibody (1:40 dilution; ab52915, Abcam), a rabbit anti-MMP-9 polyclonal antibody (1:2,000 dilution; ab38898, Abcam), a rabbit anti-Cldn1 polyclonal antibody (1:200 dilution; B6327, LSBio, Seattle, WA), and a rabbit anti-Cldn4 polyclonal antibody (1:25 dilution; B2370, LSBio) overnight at 4°C. Tissue sections were washed again in PBS and incubated with peroxidase-conjugated secondary antibodies for 30 min at 37°C. Detection of the antibody complex was done using the streptavidin-peroxidase reaction kit with 3,3′-diaminobenzidine as a chromogen (ABC kit, Vector Labs, Burlingame, CA).

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