Immunohistochemical Analysis of Cellular Biomarkers in PEG-Treated Rectal Biopsies

The biopsy sections were subjected to IHC analysis to determine the expression of several cellular biomarkers following PEG treatment. These included EGFR, Ki-67, SNAIL, Cleaved Caspase-3 and E-cadherin, all of which have previously been reported by our group to be modulated by PEG in cell culture and animal models [14 (link)]. For these studies, 2–3 rectal biopsies were formalin fixed, paraffin embedded, sectioned and subjected to the IHC evaluation by standard techniques using appropriate primary antibodies [anti-Ki67 (1:250; AbCam, Cambridge, MA), anti-EGFR (1:200; Santa Cruz, CA, anti-Cleaved Caspase 3 (1: 100; Cell Signaling Technology, Danvers, MA)], anti-SNAIL (1:100; Snai1–T18; Santa Cruz, CA) and anti-E-cadherin (1:250; Cell Signaling Technology) followed by appropriate biotinylated secondary antibodies. The antigen-antibody complexes were detected with the Vectastatin Elite ABC kit (Vector Laboratories). For negative controls, sections were processed in the absence of the primary antibodies. IHC sections were scored by the study pathologist (CW), who was blinded to the study group. A semi- quantitative scale was used to evaluate immunoreactivity of epithelial cells. The extent of staining was graded and scored as 0 (negative staining); 1+ (10% stained cells), 2+ (10–50% stained cells), and 3+ (50% stained cells).