Gtx54672

To conduct immunohistochemical analysis, deparaffinized retrieved auricular tissue sections from the first in vivo study were subjected to 0.3% H₂O₂ treatment for 20 min. After that, samples were incubated with an anti-p-NFκB p65 antibody (GTX54672, GeneTex Inc.—1:100) overnight at 4 °C. After being washed with phosphate buffered saline pH 7.4, tissue sections were then treated with the secondary antibody from the HRP Envision kit (DAKO) for an additional 20 min, followed by a second wash out and an additional 15 min of incubation with the dye diaminobenzidine (DAB). The slides were then cross stained with hematoxylin, dehydrated, and cleaned in xylene before being covered for microscopic analysis following a final PBS wash. For the purpose of determining the relative area % of immunohistochemistry expression levels of phospho-NF-κB in dermal/epidermal cellular elements, six nonoverlapping fields were arbitrarily nominated and perused from each sample^{40 ,42 (link)}.

Following the manufacturer's guidelines, 5 µm thick tissue sections, after deparaffinization, underwent antigen retrieval and were subsequently treated with 3% H₂O₂ for 20 min. They were then incubated overnight at 4°C for 30 min with anti-NF-kB p65 antibody (GTX54672, GeneTex Inc., 1:100 dilution). Afterward, sections were washed using immune washing Tris buffer and incubated with the secondary antibody HRP Envision kit (DAKO) for 20 min, followed by another wash using immune washing Tris buffer. The sections were stained with hematoxylin for 2–5 min after establishing the reaction with DAB for 2–3 minutes^{30 (link)}.

Immunohistochemical staining was conducted according to the manufacturer’s protocol. Deparaffinized antigen retrieved five microns thick tissue sections and were treated with 3% H₂O₂ for 20 min. Washed, incubated with anti p-NFκB p65 antibody (GTX54672, GeneTex Inc., Hsinchu, Taiwan—1:100) 60 min; washed by PBS, followed by incubation with secondary antibody HRP Envision kit (DAKO, Glostrup, Denmark) 20 min; washed by PBS and incubated with diaminobenzidine (DAB) for 10 min. Washed by PBS and then counterstaining with hematoxylin, the tissue section then dehydrated and clearing in xylene then the cover slipped for microscopic examination.