Ck5 xm26

BrCSCs were fixed with 2% paraformaldehyde for 30 minutes at 37°C and incubated O.N. at 4°C with the following primary antibodies: ALDH1 (44, BD), CK5 (XM26, Novocastra), CK14 (LL002, Novocastra), CK8-18 (CD10, Novocastra), MUC1 (BD Pharmigen), VIMENTIN (R28, Cell Signaling), ER (6F11, Novocastra). Thereafter, cells were labeled with secondary antibodies (Invitrogen). Nuclei were counterstained with Toto-3 (Invitrogen).

Immunohistochemical analyses were carried out on 5-µm-thick paraffin-embedded sections of breast cancer xenografts. Tissues underwent antigen retrieval, where required permeabilized with cold 0.1% Triton X 100, and stained overnight at 4 °C with antibodies against Ki67 (MIB 1, Dako), p63 (4A4, Santa Cruz), CK5 (XM26, Novocastra), CK 5/6 (D5/16B4, Dako), CK8/18 (5D3, Novocastra), Vimentin (R28), PR (16, Biocare Medical), Her2 (D8F12, CST), and ER (6F11, Novocastra). Successively, sections were incubated with biotinylated secondary antibodies and exposed to streptavidin-peroxidase (Dako). Stainings were revealed with 3-amino-9-ethylcarbazole substrate (AEC, Dako) and nuclei counterstained with aqueous hematoxylin. For H/E, tissues were stained with hematoxylin for 2 min and subsequently with eosin for 1 min.