Diaminobenzedine substrate

Immunohistochemical staining was conducted on 5 µm sections of tissue microarray (TMA) blocks as previously described.23 (link) IL17A staining was performed using a streptavidin-biotinylated horseradish peroxidase (S-ABC) kit (Novo Link Max Polymer Detection System; Novocastra) involving quenching of endogenous peroxidase activity with 3% hydrogen peroxide, blocking of nonspecific binding of antibodies (Novocastra) prior to incubation first with human IL-17 (H-132) polyclonal rabbit antibody (sc-7927) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at a dilution of 1:100 and then with biotinylated anti-rabbit immunogloublin G (Novocastra). Peroxidase was detected using diaminobenzedine substrate (Novocastra), and slides were counterstained with Mayer’s hematoxylin (Novocastra). Omission of the primary antibody was employed as a negative control. The expression of IL17A in tumor and normal samples was analyzed using the eSlide capture device (ScanScope^® CS).

Immunostaining of the heart sections for the detection of pJAK2 and pSTAT3 was performed using streptavidin-biotinylated horseradish peroxidase (Novalink Max Polymer detection system; Novocastra Laboratories, Newcastle, UK). In brief, sections were incubated in 3% hydrogen peroxide in distilled water for 5 minutes to block endogenous peroxidase activity and then washed in Tris-buffered saline (pH 7.6) for 10 minutes. Nonspecific binding of antibodies was blocked by incubation with protein block (Novocastra) for 5 minutes. Sections were incubated with rabbit polyclonal anti-pJAK2 and goat polyclonal anti-pSTAT3 primary antibodies (Santa Cruz Biotechnology Inc.) diluted 1:100 at room temperature for 1 hour. The sections were washed in Tris-buffered saline three times and incubated with biotinylated IgG (Novocastra) for 30 minutes. After washing in Tris-buffered saline and incubation with Novolink polymer (Novocastra) for 30 minutes, peroxidase was detected with diaminobenzedine substrate (Novocastra). Sections were then washed in distilled water, stained with Mayer’s hematoxylin, and mounted in DPX. The same procedure, with the omission of incubation in primary antibodies, was followed for negative control sections.