Figure 3 Expression of Fas ligand (FasL) at the luminal surface of normal colonic epithelium coincides with increased frequency of terminal transferase mediated dUTP nick end labelling (TUNEL) positive epithelial cells. (A) Immunoperoxidase staining using a FasL specific rabbit polyclonal antibody was performed on paraffin wax embedded normal colonic sections. Slides were counterstained with haematoxylin. FasL positive immunohistochemical staining (brown) is shown in a representative normal colonic specimen. FasL expression was detected in luminal colonic epithelium (arrow). As a control for specificity of antibody detection, the FasL immunising peptide was included during primary antibody incubation. Competitive displacement of staining by the immunising peptide confirmed FasL specificity (not shown). (B) FasL immunophosphatase (alkaline phosphatase conjugated anti-alkaline phosphatase; APAAP) staining (red) was performed using a FasL specific monoclonal antibody. Slides were counterstained with haematoxylin. Results from staining with the FasL specific monoclonal antibody agreed with those obtained using the polyclonal antibody—FasL positive epithelial cells (red; arrow) were present at the luminal surface. (C) Immunoperoxidase staining with the same FasL specific monoclonal antibody as used in (B) gave identical results. Thus, staining was independent of secondary detection systems because similar results were obtained using either APAAP or immunoperoxidase assays. (D) FasL positive luminal epithelium (brown) from (C) illustrated at higher magnification. (E) Negative crypt epithelium from (C) illustrated at higher magnification. (F) Cell death detection in situ by TUNEL. Cell death was detected by enzymic labelling of DNA strand breaks using TUNEL in normal colonic sections sequential to those shown in (A). Only those cells with positive TUNEL staining (brown) and exhibiting apoptotic morphology (arrow) were considered to be apoptotic. Control sections (not shown) where the terminal transferase was omitted were negative. An increased frequency of TUNEL positive epithelial cells was detected at the luminal surface, where cell death was coincident with FasL expression. These results are representative of 20 normal colonic specimens.