Df1485

TTF-1 and CK7 IHC was carried out on formalin-fixed, paraffin-embedded tissue 4 μm sections using anti-TTF-1 (mouse, 8GTG3/1, 1:200, Dako, Carpinteria, CA, USA) and anti-CK7 (mouse, OV-TL 12/30, 1:1000, Dako). Antibody incubation and detection were carried out at 37°C on a Menarini intelliPATH FLX (A. Menarini Diagnostics, UK) using Menarini's reagent buffer and detection kits unless otherwise noted. Antigen retrieval was carried out in a pressure cooker in citrate buffer pH6 for 2 min at 123°C. Pan-CK, CD44 and vimentin IHC was carried out on formalin-fixed, paraffin-embedded tissue 4 μm sections using anti pan-cytokeratin antibody (mouse, M3515, 1:60, ER1 20 min Dako), anti CD44 (mouse, DF1485, 1:100, ER2 10 min, Dako) and anti-Vimentin (mouse, V9, CC1 32 min, Roche). Pan-CK and CD44 staining was carried out on the LEICA Bond Max Platform and Vimentin staining on the Ventana Discovery Ultra platform (Roche). Appropriate positive, negative and isotype controls were included with the study sections (not shown). Digital images of whole-tissue sections acquired using a Leica SCN400 histology scanner (Leica Microsystems).

Tissue samples for light microscopy were fixed in 4% formaldehyde and embedded in paraffin using routine procedures. Five-micrometer thick sections were cut from the tissue blocks and stained with hematoxylin-eosin.
For immunohistochemical staining the following primary antibodies were used: CD133/1 (AC133, 1 : 100, Miltenyi Biotec, Bergisch Gladbach, Germany) and CD44 (DF1485, 1 : 100, Dako, Glostrup, Denmark). No special pretreatment was used. The primary antibodies were visualized using the supersensitive streptavidin-biotin-peroxidase complex (Biogenex, San Ramon, CA). Appropriate positive and negative control slides were employed.