Mammary gland whole-mount staining and IHC

X-gal staining and whole mounting of mammary glands was carried out as previously described1 (link)7 (link)13 (link). #3 and #8 thoracic glands from the recipient nude mice were used as negative staining controls for all X-gal staining procedures. X-gal-stained whole mounts were embedded in paraffin, sectioned at 6.0 μm, and counterstained with nuclear fast red (Sigma-Aldrich, St Louis, MO, USA). Primary antibodies used were rabbit-anti-Cytokeratin 8 (ab53280 1:100; Abcam, Cambridge, MA, USA), rabbit-anti-keratin wide spectrum (Z0622 1:500; Dako, Carpinteria, CA, USA), rabbit-anti-ER alpha (sc-542 1:75; Santa Cruz Biotechnology, USA), rabbit-anti-progesterone receptor (A0098 1:150; Dako), and mouse-anti-SMA 1A4 (1:100; Life Technologies, Carlsbad, CA, USA), anti-alpha-lactalbumin31 (link) and anti-caseins32 (link). IHC staining procedure was carried out using the RTU Vectastain Universal ABC Kit and DAB peroxidase substrate kits (Vector Laboratories, Burlingame, CA, USA) per the manufacturers protocol. All sections were counter-stained with nuclear fast red or haematoxylin.