Immunocytochemical Analysis of Epithelial Cells

To evaluate the expression of cytokeratin 8/18, a selective marker of epithelial cells, TECs (at passage 2–4) were cultured on 12-mm diameter coverslips until reaching semi-confluence, when they were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA). Permeabilization of cell membranes and blocking of unspecific binding sites were performed by incubation with PBS containing 0.1% Triton and 0.2% bovine serum albumin (both from Sigma-Adrich, Milan, Italy). TECs were then incubated overnight at 4°C with a mouse monoclonal antibody anti-Ck 8/18 (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), diluted 1∶200 in PBS containing 2% human serum and 2% normal goat serum. After being washed, cells were incubated for 180 minutes at room temperature with an anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Sigma-Aldrich, Milan, Italy). The chromogen reaction was developed using a DAB substrate for Peroxidase. For control purposes, the primary antibody was replaced by a non-immune normal goat serum. The observations were documented using light microscope Leica DMR equipped with Leica DC200/400 camera. Image acquisitions were all performed by the same observer (ASB). This experiment was performed on all thymi from which cell cultures were obtained (n = 10).