Anti er α sc 542

Mammary glands and tumors were collected, fixed with 10% formalin and embedded in paraffin using standard procedures. Sections (5 μm) were mounted onto slides and stained with hematoxylin and eosin to be evaluated by pathologists. Antibodies used for immunostaining were: anti-ER-α (sc-542, Santa Cruz, CA, USA), anti-HER2 (#4290, Cell Signaling Technology), anti-PGR (NCL-L-PGR-312, Dako, CA, USA), anti-Cytokeratin 5 (PRB-160P, Covance, Princeton, NJ, USA), anti-Cytokeratin 8 (10R-C177AX, Fitzgerald Industries International, Acton, MA, USA), anti-Cytokeratin 14 (E2624, Panomics, Fremont, CA, USA), and anti-Cytokeratin 10 (PRB-159P, Covance). Immunostaining was performed with standard protocols using Bond™ Polymer Refine Detection (Leica Biosystem, Buffalo Grove, IL, USA) according to the manufacturer’s instructions.

Proteins (30 μg) were extracted from the hippocampus, separated on a 7.5% polyacrylamide gel and transferred onto nitrocellulose membranes as previously described [33 (link)]. The blots were probed overnight at 4°C with polyclonal rabbit (1/400 anti-AR #sc-816 and 1/400 anti-ERα #sc-542; Santa-Cruz Biotechnology) or monoclonal mouse antisera (1/10000 anti-glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) #sc-32233 from Santa-Cruz Biotechnology). They were then incubated with horseradish peroxidase-conjugated secondary anti-rabbit #111.035.144 or anti-mouse #115.035.062 antisera (1/5000, Jackson Immunoresearch). Signals were visualized with pico- (for GAPDH) and femto- (for AR and ERα) Super Signal detection kit (Thermo Scientific), quantified with ImageJ software (NIH) and normalized with respect to GAPDH. As we have previously shown by immunohistochemistry [33 (link)], AR protein was detected in the hippocampus of control (AR^fl/Y) males but not in their mutant (AR^NesCre) littermates (Fig 1A). By contrast, hippocampal ERα protein was present in both AR^fl/Y and AR^NesCre males (Fig 1B). Quantification of ERα protein normalized to GAPDH showed similar amounts in the two genotypes (Fig 1C).