Quantifying Apoptosis in Jejunum Tissue

Apoptotic cells were quantified in the jejunum using two independent but complementary techniques: active caspase-3 staining and morphologic analysis of H&E-stained sections [27] (link). For caspase-3 staining, sections were deparaffinized, rehydrated, and incubated in 3% hydrogen peroxide for 10 minutes. Sections were then placed in Antigen Decloaker (Biocare Medical, Concord, CA) and heated in a pressure cooker for 45 minutes. After sections were blocked with 20% normal goat serum (Vector Laboratories, Burlingame, CA), they were incubated overnight with rabbit polyclonal anti-active caspase-3 (1∶100; Cell Signaling, Beverly, MA) at 4°C. The following day, sections were incubated with goat anti-rabbit biotinylated secondary antibody (1∶200; Accurate Chemical and Scientific, Westbury, NJ) for 1 hour followed by horseradish peroxidase (HRP)- labeled streptavidin (Accurate Chemical and Scientific) for 1 hour. Sections were then developed with diaminobenzidine and counterstained with hematoxylin.
Apoptotic cells were identified on H&E-stained sections via characteristic morphological changes including cell shrinkage with condensed and fragmented nuclei and then quantified in 100 well-oriented contiguous crypt-villus units.