Visualization of Cell Death in Colonic Tissue

TUNEL staining was done by the In Situ Cell Death Detection Kit^® (A23210, Roche, Mannheim, Germany) for the visualization of cell death in colonic tissue, according to the method described earlier [50 (link)]. Paraffin slides containing colonic sections were deparaffinized and rehydrated by xylene and ethanol. These sections were washed and then post-fixed using ethanol and acetic acid (2:1) solution. These sections were then treated by proteinase K (100 μg/mL), treated in 3% H₂O₂, permeabilized with 0.5% Triton X-100, and treated with TUNEL reaction mixture. After washing, these sections were visualized by Converter-POD with 0.03% 3,3′-diaminobenzidine. After counterstaining with Mayer hematoxylin (DAKO), these sections were mounted on gelatin-coated slides, air-dried overnight at room temperature, and next coverslipped by Permount^® (Thermo Fisher Scientific).