IHC was performed twice on a total of 12 sample blocks, one from each uterine horn of six animals (three controls and three treated). Two sections, one from a control animal the other from an IFN-treated animal, were mounted on each precoated slide. Sections were air dried, incubated at 60°C for 30 min, and kept at 4°C until used. Immunostaining procedures were performed at room temperature unless otherwise noted. First, slides were deparaffinized in xylene and washed with ethanol. Endogenous peroxidase activity was then quenched with 3% H2O2 in methanol for 20 min.
After rehydratation, sections were incubated in 0.1 M citrate buffer for 15 min at 95°C but only for GM-CSF immunostaining (this step was not necessary to detect both COXs). The blocking, antibody, and ABC steps were the same as for experiment 1 except for the antibody concentrations (anti-GM-CSF 4 |xg/ml; anti-COX 1/2000). AEC was left to reveal for 15 (COXs) or 30 (GM-CSF) min. Mayer hematoxylin was used for counterstaining, and aqueous mounting medium was applied on the sections.
Slides were observed under a Zeiss Axioskop2 Plus microscope (Toronto, ON, Canada) linked to a digital camera from Diagnostics Instruments (Sterling Heights, MI). Images were captured by the Spot software (Diagnostics Instruments) and analyzed with Image-Pro Plus from Media Cybernetics (Silver Springs, MD).