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The 5 paired ADOL tissue samples were formalin-fixed, paraffin-embedded, and sectioned. Immunohistochemical stains were performed with either a monoclonal mouse anti-human estrogen receptor alpha (ER-α) antibody [6F11] (1:50 dilution, BioRad, Hercules, CA) or a monoclonal mouse anti-human CD68 antibody (1:200 dilution, Dako, Carpenteria, CA). ER-α staining was performed using the ImmPRESS anti-mouse IgG horseradish peroxidase (HRP) polymer (Vector Laboratories, Burlingame, CA), and CD68 staining was performed using a standard avidin-biotin-peroxidase technique. Antigen-antibody complexes were visualized using 3-diaminobenzidine (DAB) chromagen (DakoCytomation, Carpenteria, CA). Slides were scanned at 40× using the Aperio® AT2 Digital Whole Slide Scanner (Leica Biosystems, Buffalo Grove, IL) and visualized using the Aperio® ImageScope v. 12.4.0.7018 (Leica Biosystems).

Formalin-fixed, paraffin-embedded mouse mammary glands (PND2 glands collected from fertility study) were sectioned and stained using standard H&E staining protocol. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) immunohistochemical analysis was done to detect apoptosis. Formalin -fixed, paraffin-embedded mouse mammary glands (PND2 glands collected from fertility study) were stained with ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit (Cat# S7101, Millipore, Billerica, MA) using the manufacturer’s recommendations. Staining was visualized using 3-diaminobenzidine (DAB) chromagen (DakoCytomation, Carpenteria, CA) and counterstained with hematoxylin. The slides were dehydrated through graded ethanol, cleared in xylene, and coverslipped.