Horseradish peroxidase conjugated goat anti mouse igg secondary antibody

Duodenum, jejunum, and ileum were fixed in 4% paraformaldehyde for 24–48 h at room temperature and the histopathology and immunohistochemistry were performed according to our previous reported method (82 (link)). The detection of PDCoV antigens was performed using anti-PDCoV-N protein-specific monoclonal antibody (prepared in our lab), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich).

Culture supernatants from cells treated with mAb plus adiponectin were analyzed with IL-6 and IL-8 ELISA kits (BD Bioscience Korea, Seoul, Korea) in accordance with the protocol of the manufacturer. To screen hybridoma clones for mAbs against adiponectin, 96-well plates were coated with adiponectin (200 ng/well), hybridoma culture supernatants were added after blocking, wells were incubated with a 1:1,000 dilution of horseradish peroxidase–conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich Korea), and the ELISA procedure was completed. Sera from CIA were obtained from heart puncture and analyzed for adiponectin, IL-6, TNF-α, and RANKL by using the Luminex^® 200™ Total System (Luminex Corporation, Austin, TX, USA) as previously described [33 (link)].