Immunohistochemical Analysis of NF-κB Expression

Immunohistochemistry was used to evaluate the expression pattern of NF-κB protein as per the method previously explained [50 (link),51 (link)]. In brief, antigen retrieval was performed to unmask the antigen sites. The primary monoclonal antibodies against NF-κB were added to lung sections and incubated overnight at 4 °C temperature. Biotinylated secondary antibodies (Abcam kits, Cambridge, UK) were added for 45 min at room temperature. Thereafter, horseradish peroxidase (HRP)-conjugated streptavidin was applied for a further 35 min at room temperature. Diaminobenzidine (DAB) was used as chromogen and haematoxylin stain was used as a counterstain. The images were captured by a digital camera, and the results were interpreted accordingly.