Vimentin v9

Tumour tissue and the agarose-embedded close-to-patient cancer cells were formalin fixed and paraffin embedded (FFPE) before 4 μm sections were cut for both H&E and immunohistochemistry (IHC) analysis. IHC was performed using standard techniques and in line with the manufacturer's instructions for the following primary antibodies: Cytokeratin (MNF116, DAKO), EpCam (Ber-EP4, DAKO), CD44 (DF1485, DAKO), p53 (DO-7, DAKO), Vimentin (V9, DAKO), TFF3 (Abcam), and ALDH1A1 (EP1933Y, Abcam) (see online supplementary method S6). Sections were viewed with a Leica DMLB Bright-field Microscope (Leica-microsystems, Milton Keynes, UK) and images acquired with Leica QWin Standard v3 software. The presence of any characteristically stained cells was considered positive with respect to negative controls, and confirmed by a second blinded individual.

Staining procedures were performed as previously described [55 (link)]. For human vimentin (VimentinV9, M0725, Dako, 1:1000) and cleaved caspase-3 staining (ASP175, Cell Signaling Tech, 1:400), heat-induced epitope retrieval was performed by microwave boiling for 15 min in TE (Tris/EDTA, pH 9) buffer (Dako). Collagen staining was performed using Sirius Red stain and analyzed by polarized light microscopy.

Subcutaneous nodules on mouse backs were fixed in 20% formalin and embedded in paraffin. Cut paraffin sections were deparaffinized, dehydrated, and treated with 2% proteinase K (Dako) in Tris–HCl buffer solution (pH 7.5) for 5 min at room temperature, or heated in ChemMate Target Retried Solution (Dako) for 5–20 min in a high-pressure steam sterilizer for epitope unmasking. After washing with distilled water, samples were placed in 1% hydrogen peroxide/methanol for 15 min to block endogenous peroxidase. The sections were then incubated at room temperature for 60 min in primary antibodies diluted with antibody diluent (Dako). The following primary antibodies against various human differentiation antigens were used: vimentin (V9, M0725, Dako, Glostrup, Denmark), albumin (Dako), AE1/AE3 (712811, NICHIREI), and Ki67 (ABCAM, ab15580). Then, they were washed three times with 0.01 M Tris buffered saline (TBS) solution (pH 7.4) and incubated with goat anti-mouse or anti-rabbit immunoglobulin labeled with dextran molecules and horseradish peroxidase (EnVision, Dako) at room temperature for 30 min. After three times washes with TBS, they were incubated in 3,3′-diaminobenzidine in substrate-chromogen solution (Dako) for 5–10 min. Negative controls were performed by omitting the primary antibody. The sections were counterstained with hematoxylin.