Tunel apoptosis assay kit

Intestinal tissue was fixed in 4% paraformaldehyde for 48 h at room temperature (~20˚C), embedded in paraffin, and cut into 5-µm sections. A TUNEL apoptosis assay kit (Sigma-Aldrich; Merck KGaA) was used according to the manufacturer's protocol. After dewaxing, hydration and cell permeabilization using 0.2% Triton X-100 (ZSGB-BI; cat. no. ZLI-9308), TUNEL reaction solution, converter-peroxidase, and 3,3'-diaminobenzidine (DAB; ZSGB-BIO; cat. no. ZLI-9018) were added dropwise in sequence. At room temperature, 100 µl DAB substrate was added dropwise to the tissue on the glass coverslip for color development. After dropping, the samples were observed under the microscope, and the color development was stopped when the appropriate amount of yellowish-brown appeared. The stained cells appeared as if the chromatin was condensed, marginalized and divided into blocks (apoptotic bodies), and the nuclear membrane was cracked. After sealing with neutral balsam, the samples were mounted under glass coverslip with glycerol and analyzed under light microscope (magnification, x200). Five fields of view were randomly selected from each tissue and analyzed separately by three professional pathology teachers.

Apoptosis of epithelial cells was analyzed using the Terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and the number of apoptotic cells was quantified. Intestinal tissues were fixed, embedded in paraffin wax, and cut into 5-µm sections. The TUNEL apoptosis assay kit (Sigma-Aldrich; Merck KGaA) was used for the experiments according to the manufacturer's instructions. The apoptotic cells stained with brown nuclei were analyzed under an optical microscope (magnification, ×400).