Inhibin βa hpa020031

Assembly and IHC staining of human breast tissue microarrays (TMAs) were performed by the Biorepository and Tissue Analysis core at MUSC. TMAs consisted of matched normal and tumor tissue as well as normal and metastatic lymph node biopsies from patients for which survival data was available (Supplementary Table S2). For IHC staining of formalin-fixed, paraffin-embedded TMAs and mouse lung tissue, sections were deparaffinized in xylene, rehydrated in alcohol, and processed as follows: The sections were incubated with target retrieval solution (S2367; Dako, Carpinteria, CA, USA) in a steamer for 45 min followed by 3% hydrogen peroxide solution for 10 min and protein block (X0909; Dako) for 20 min at room temperature. Sections were incubated overnight in a humid chamber at 4°C with antibodies against Inhibin βA [HPA020031] (1:3200; Sigma), E-cadherin [#3195], Vimentin [#5741] or Ki-67 [#12202] (1:400; Cell Signaling) followed by biotinylated secondary antibody (PK6106; Vector laboratories, Burlingame, CA, USA) for 30 min and ABC reagent for 30 min. Immunocomplexes of horseradish peroxidase were visualized by DAB reaction (K3468; Dako), and sections were counterstained with hematoxylin before mounting. Micrographs of stained sections were taken using an Olympus DP72 8-bit RGB camera with Cellsens acquisition software.