If488 tyramide

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).

After fixed in 4% paraformaldehyde for 7 days, lung tissues were embedded in paraffin, cut into slices. After hematoxylin and eosin (H&E) staining, pathological changes were evaluate and scored on a 0–5 severity scale, according to the thickened alveolar walls, cell aggregation, blocked bronchioles and lung consolidation. Infiltration was also evaluated and scored on a 1–4 severity scale.
For immuno-fluorescence assay, the sections were deparaffinized and rehydrated, followed by Citrate-mediated antigen retrieval. After blocking, the primary antibody Anti-Syndecan-1/CD138 Rabbit pAb (cat. no. GB115052, Servicebio), HRP-conjugated goat-anti-rabbit IgG and CY3-Tyramide (cat. no. G1223, Servicebio) were used, followed by Citrate-mediated antigen retrieval. Then another primary antibody Anti-IL-17 Rabbit pAb (cat. no. GB11110-1, Servicebio), secondary antibody HRP-conjugated goat-anti-rabbit IgG and the corresponding iF488-Tyramide (cat. no. G1231, Servicebio) were used. Cell nuclei were stained using 49, 6-diamino-2-phenylindole (DAPI) (cat. no. G1012, Servicebio). The slide scanner Pannoramic MIDI (3DHISTECH) was utilized to image whole slide.