Ab97389

Antigen retrieval was performed by treating CEA sample sections with 1 mM ETDA at 95°C for 10 min followed by 15 min in 1% H₂O₂ in PBS to quench endogenous peroxidase activity. Samples were then blocked with 2% goat serum in PBS for 20 min at room temperature and incubated with a primary anti-human NPR-C antibody (ab97389, Abcam, Cambridge, MA) diluted 1:1000 in PBS containing 0.05% Tween 20 and 1% goat serum overnight at 4°C. Avidin/Biotin blocking was incorporated into the serum block and primary antibody steps following manufacturer instructions (Vector Laboratories, Burlingame, CA). The sections were then incubated with a biotinylated goat anti-rabbit secondary antibody at a 1:200 dilution (Vector Laboratories, Burlingame, CA) in PBS containing 0.05% Tween 20 for 30 min followed by ABC reagent (Vector Laboratories, Burlingame, CA) for 30 min. Vector DAB substrate was used for staining sections for 60 sec. Control sections with only 1% goat serum in place of the primary antibody were run concurrently with the NPR-C stained slides. An additional negative control using polyclonal rabbit IgG antibody diluted to the same concentration as the diluted rabbit anti-human NPR-C primary antibody (1 μg/ml) was also used.