Immunohistochemical Analysis of Apoptotic Markers

Immunohistochemistry for cleaved caspase-3 and caspase-9 was done according to the method described earlier [51 (link)]. Paraffin slides containing colonic sections were deparaffinized by xylene and ethanol and rehydrated. These sections were washed, and after boiling 10 mM citric acid (pH 6.0) for 10 min, these sections denatured. These sections were treated with mouse anti-cleaved caspase-3 (9661S) and anti-cleaved caspase-9 antibodies (9507S, Cell Signaling Technology Inc., Danvers, MA, USA) diluted 1:200 overnight. These sections were treated with biotinylated anti-rabbit (BA-1000) and anti-mouse secondary antibodies (BA-2000, Vector Laboratories, Burlingame, CA, USA) for 1 h. These sections were then treated with avidin-biotin-peroxidase complex (PK4010, Vector Laboratories) at room temperature for 1 h. By treating these sections with the solution containing 0.05% 3,3-diaminobenzidine and 0.01% H₂O₂ in 50 mM Tris-buffer (pH 7.6) for approximately 3 min, immunoreactivity was visualized. After counterstaining with Mayer hematoxylin (DAKO), these sections were mounted on gelatin-coated slides, air-dried overnight at room temperature, and coverslipped by Permount^® (Thermo Fisher Scientific).