Sections were air dried and fixed with 4% paraformaldehyde in PBS (pH 10) for 30 min. Immunostaining procedures were immediately performed at room temperature unless otherwise noted. First, endogenous peroxidase activity was quenched with 3% H2O2 in PBS for 15 min. Nonspecific binding sites were then blocked with 10% goat serum in PBS for 1 h.
Antibodies were diluted in 0.5% goat serum in PBS (1/4000 for anti-COX sera or nonimmune serum; 1 |xg/ml for GM-CSF-specific antibody or IgG1 control) and applied overnight at 4°C. Sections were subsequently incubated with biotinylated secondary antibody (goat-anti-rabbit for COXs, goat-anti-mouse for GM-CSF) for 40 min and with ABC (Vector Labor-atorie, Burlington, ON, Canada) reagent (Elite for GM-CSF) for 30 min. Immunostaining was revealed by using 3-amino-9-ethylcarbazole (AEC) for 12 min. Harris hematoxylin was used for counterstaining, and aqueous mounting medium (Fisher Scientific) was applied on the sections.
In experiment 2, cross-sections (4 |xm) were prepared from paraffin-embedded uteri with a microtome.