Quantifying TLR9 and NF-κB in Human NP

To assess the levels of TLR9 and NF-κB in human NP specimens, the sections were stained using fluorescent multiplex immunohistochemistry [26 (link)]. The sections were dewaxed and rehydrated with xylene and gradient ethanol, then subjected to antigen repair with pepsin for 30 min at 37 °C, followed by serum blocking for 30 min at 21 °C, and incubation with primary overnight at 4 °C and secondary antibodies for 50 min at 21 °C. Antibodies were coupled with tyramide-conjugated fluorophores as follows: TLR9 with iF488-Tyramide (G1231, Servicebio) and NF-κB with iF555-Tyramide (G1233, Servicebio). The immunolabeled sections were imaged using a fluorescence microscope (Eclipse 80i, Nikon).