Paraffin-embedded blocks were sectioned (5 µm thickness) and transferred to silanized glass slides. The sections were then deparaffinised in xylene and rehydrated in a graded series of alcohol. Antigen retrieval was fulfilled by heating the sample in 0.01 M citrate buffer (pH 6.0) using a microwave vacuum histoprocessor (RHS-1; Milestone, Bergamo, Italy) at a final temperature of 121°C for 15 min. To block endogenous peroxidase activity, sections were blocked with 3% hydrogen peroxide in methanol for 10 min. Slides were incubated with mouse anti-CEACAM-1/CD66a (R&D Systems), anti-EpCAM (clone: MOC-31, Abcam), anti-CD45 (Abcam), and anti-CD56 (Abcam) antibodies, which were diluted to a ratio of 1:50 in Dako antibody diluent (Dako, Carpinteria, CA, USA) with background-reducing components at room temperature for 30 min. After washing, the Dako EnVision Plus system (Dako) was used at room temperature for 5 min. The immunoreaction was performed with diaminobenzidine for 5 min, followed by hematoxylin counterstaining. Immunofluorescence staining with confocal microscopy was performed as previously described with anti-CEACAM1 and anti-EpCAM antibodies.29 (link)