Slides (5 µm thick) were autoclaved in Target Retrieval Solution (Dako, S1699, Denmark), incubated in Proteinase K (S3004, Dako, Denmark) or with EDTA- buffer (Prosan, Belgium) according to the immunolabelling for Ki67, CD31, and FITC-dextran, respectively. Endogenous peroxidases were blocked by 3% H2O2/H2O (Merck, Belgium) for 20 minutes, and nonspecific binding was prevented by incubation in PBS/Bovine Serum Albumin 10% (Fraction V, Acros Organics, NJ). Tumor sections were incubated with a mouse monoclonal anti-human Ki-67 antibody (1/100) (clone MIB-1, M7240; DAKO, Denmark), a rat anti-CD31 antibody (1/100) (Ab56299, Abcam, United Kingdom), or a ready-to-use anti-fluorescein antibody (Converter-POD, Roche, France). After 3 washes in PBS or Tris-HCl for CD-31 staining, slides were incubated with an HRP-conjugated secondary antibody, after post antibody blocking (DPVB Blocking, Immunologic NL) for pimonidazole staining, and revealed with Vector DAB (SK-4100, Vector Laboratories, Burlingame, CA, USA). Slides were counterstained with hematoxylin. For lung metastases quantification, six lung sections of 5 µm, spaced by 10 sections of 5 µm, were immunostained with an antibody against human Ki67 as previously described (6 (link)). Metastases were manually counted and classified according to their size (<10 cells, 10 to 50 cells, 50 to 100 cells, >100 cells).
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