Mouse anti human ck18

Cells (1×10⁵)were resuspended in 100 μl phosphate-buffered saline (PBS) supplemented with 0.5% BSA and 2 mM EDTA, and incubated with 10 μl polyclonal mouse anti-human CD133-PE conjugated antibody (1:100; Miltenyi Biotec, Inc.), monoclonal mouse anti-human cytokeratin (CK)8 (1:100) and mouse anti-human CK18 (1:100; Dako, Glostrup, Denmark) for 10 min at 4°C. Following washing with PBS, the cells were resuspended in a solution of PBS and 2 μl 7-amino-actinomycin D (7-AAD), and analyzed using a EPICS^® XL™ flow cytometer (Beckman Coulter).

Paraffin blocks were sectioned at approximately 3–5 microns thickness. Sections were placed on SuperFrost Plus™ glass slides. Slides were incubated overnight at 64 °C. Slides were stained using the standard procedure in Ventana BenchMark Ultra automated slide stainer in combination with Ventana UltraView Universal DAB Detection Kit (Ventana, Roche Diagnostics cat #760-500). The slides were stained with the following antibodies: monoclonal mouse anti-Human Ki-67, clone MIB-1 (with Nuclear Dekloaker Dako as heat mediated antigen retrieval solution, cat# M7240), diluted 1:200, mouse anti-Human E-cadherin (with Nuclear Dekloaker Dako as heat mediated antigen retrieval solution Invitrogen, cat# #33-400, diluted 1:50, mouse anti-Human CK-7(with citrate as heat mediated antigen retrieval solution, Dako, cat#7019), diluted 1:200, mouse anti-Human CK-18 (with citrate as heat mediated antigen retrieval solution, Dako, cat#7010), diluted 1:3000, mouse anti-Human GATA-3 (with citrate as heat mediated antigen retrieval solution, Zytomed, cat#BMS054), ready to use.