Anti med12

Tissue cores were collected from tumors and myometrial controls of each case for tissue microarray and represented in duplicate. Antibodies used for this study included HMGA2 (1:500, BioChem Inc., CA) and Anti-MED12 (1:200, Proteintech Europe, Manchester, UK). Tissue microarrays were sectioned 4 m in thickness. After deparaffinization and antigen retrieval, all immunohistochemical staining was performed on a Ventana Nexus automated system (Tucson, Arizona). In brief, endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Primary antibodies were detected using standard biotinylated anti-mouse or anti-rabbit secondary antibodies. Semiquantative immunointensity was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong) and percentage was scored as 0 (0%), 1 (≤10%), 2 (11–50%), 3 (51–80%) and 4 (>80%). Immunoreactivity for MED12 was scored for intensity only as it is diffusely expressed in all positive cases. The majority of HMGA2 positive cases showed strong and diffuse immunoreactivity for HMGA2 and only those cases with weak or faint immunoreactivity (intensity score 1) for HMGA2 showed a patchy staining pattern. Therefore, a combined intensity and percentage of immunoreactivity for HMGA2 were used. Moderate to high immunoreactivity (≥2) for HMGA2 in >50% of nuclei was read as HMGA2 positive.

Immunofluorescence was performed as previously described^{19 (link)}. Briefly, human myometrium and leiomyoma tissues were fixed in modified Davidson’s solution (Electron Microscopy Science, Hatfield, PA), embedded in paraffin, and sectioned at 5 µm. Anti-MED12 (1:200, Proteintech Europe, Manchester, UK), αSMA (1:50, Abcam, Cambridge, MA) were used as primary antibodies. Alexa Fluro 488 goat anti-mouse and Alexa Fluro 594 goat anti-rabbit (Molecular Probes/Invitrogen) were used as secondary antibodies. For the counter staining, sections were incubated with 150 ng/ml DAPI (4’,6-diamidino-2-phenylindole dihydrochloride) (Sigma-Aldrich) for 5 min at room temperature. Confocal images were obtained with an Olympus Fluoview FV10i Confocal Microscope system (Olympus America Inc., Center Valley, PA).