Immunohistochemical Protein Expression Analysis

Immunohistochemical staining was achieved according to our previously published report [18 (link)]. Briefly, after the deparaffinization protocol described above, slides were treated with proteinase K to clear formaldehyde remains from the antigen epitope and then washed. A diluted H₂O₂ solution (3% in methanol) was employed for quenching peroxidase reactivity. After washing, slides were treated with a normal serum-like (NGS). Slides were then incubated for a whole night with primary antibodies, as demonstrated in material sections with a dilution factor of 1:100. The next morning, slides were consecutively treated with a biotin-tagged 2° antibody (dilution 1:50) and with an ABC kit (Santa Cruz Biotechnology) and then stained in DAB solution. This step was followed by gradient ethanolic dehydration and fixation in absolute xylene (reverse deparaffinization protocol) and mounting with a glass coverslip. By using an Olympus light microscope at 40× magnification scale, slides were analyzed for hyperactivated p-JNK, TNF-α, caspase-3, and COX2 using an ImageJ program [19 (link)].