Publication protocol
Tissue was embedded in paraffin, cut into 5-μm sections, and mounted onto slides. Immunohistochemical analysis of PR expression was performed as previously described (15–17). Briefly, slides were deparaffinized and dehydrated through a series of xylene and ethanol washes. After a 5-minute rinse in distilled water, slides were steamed in 0.01 M sodium citrate buffer for 15 minutes and cooled for 45 minutes. Slides were rinsed for 5 minutes in PBS with 0.1% Tween 20 (PBST), and sections were circumscribed with a hydrophobic pen. Endogenous peroxidase was quenched with 3% hydrogen peroxide for 5 minutes followed by a 5-minute PBST wash. Nonspecific binding was blocked with 5% normal goat serum in PBST for 1 hour at room temperature. The primary antibody used was PR H-190 (sc _7208; 1:800) and was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Slides were incubated with the primary antibody overnight at 4°C. Normal goat IgG (Santa Cruz Biotechnology) was used as a negative control; normal day-14 endometrium was used as a positive control. Goat antirabbit biotinylated secondary antibody was used for PR (Vector Laboratories, Burlingame, CA) and applied for 1 hour at room temperature. Slides were washed in 1× PBS, incubated in ABC Elite (Vector Laboratories) for 30 minutes at room temperature, washed in 1× PBS, and incubated for 41 seconds with diaminobenzidine (Vector Laboratories). A 30-second exposure to hematoxylin was used as a counterstain. Slides were rehydrated through 5-minute ethanol and xylene washes and mounted with Permount (Thermo Fisher Scientific, Waltham, MA).
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