Sc 538

A midsection (between the fimbrial end and cervical end) from a fixed uterine horn was embedded in paraffin and sectioned at 5 mm. Sections were either stained with hematoxylin and eosin (H&E) for histopathological analyses or used for immunohistochemistry. Immunostaining was performed as previously described in Gao et al. (2014) (link). The antibodies used were ERa (sc-542, Santa Cruz Biotechnology, 1:200 dilution), progesterone receptor (PR; sc-538, Santa Cruz Biotechnology, 1:100), PTEN (138G6, Cell Signaling, 1:50), AKT (11E7, Cell Signaling, 1:50), P-AKT (D9E, Cell Signaling, Danvers, MA, USA, 1:50), and p27 (sc-528, Santa Cruz Biotechnology, 1:50). The immunopositivity and immunointensity of ERa were assessed by the H score as previously described (McNamara et al. 2013) (link). Different compartments of uterus were assessed separately: glandular epithelial cells, luminal epithelial cells, stroma, and myometrium. In brief, the H score was obtained by assessing immunointensity (scales of 0-3) and prevalence in 100 cells over five different areas in stroma and myometrium and prevalence in 20 cells over five different areas in glandular and luminal epithelial cells. All slides were counted twice to assess inter-observer variability.