Anti wap

Dissected mammary fat pads were fixed in MethaCarn and embedded in paraffin. Seven μm-thick sections were deparaffinized before staining with primary antibodies (overnight, 4°C), and secondary antibodies (1 h, room temperature). Nuclei were counterstained with DAPI. Primary antibodies used were: rabbit polyclonal anti-PAR3 (1:200; Chemicon), anti-aPKC (1:200; clone C-20, Santa Cruz Biotechnology), anti-RAB11A (1:200; Abcam), anti-pSTAT5 (Tyr694, 1:100; Cell Signalling), anti-cleaved caspase 3 (1:100; Cell Signalling), anti-WAP (1:300; clone R-131, Santa Cruz Biotechnology) and anti-keratin 5 (K5) (1:2,000; Covance); rabbit monoclonal anti-KI67 (1:100; clone SP6, Neo Markers); and mouse monoclonal anti-HTT (1:300; 4C8), anti-E-cadherin (1:200; BD Bioscience) and anti-GM130 (1:100; BD Bioscience). Antigen retrieval was performed by boiling the slides for 10 min in a microwave in 10 mM citrate buffer (pH 6) for cleaved caspase 3, Ki67, WAP, and p-STAT5A, or in EDTA buffer (pH 8.8) for 10 min for PAR3, aPKC, RAB11A, HTT, GM130, K5, and E-cadherin antibodies. Secondary antibodies used were goat anti-mouse and anti-rabbit conjugated to AlexaFluor-488 or AlexaFluor-555 or Biotin (Vector Laboratories).