Apoptag

Cell death was analyzed by TUNEL assay following the protocol from the manufacturer (ApopTag, S7101, Merck-Millipore or In situ cell death detection kit, Fluoroscein, ROCHE). Apoptotic cells were counted from Confocal (Leica SP5) of at least three-eight distinct fields of each well per group from 2–5 different experiments. Cells were counted using ImageJ software (NIH, Bethesda, MD, USA).

Cell death was investigated using a TdT-mediated dUTP nick end-labeling (TUNEL) assay according to the manufacturer’s protocol (ApopTag, S7101, Merck, Millipore). Four micron-thick sections were prepared from paraffin-embedded jejunum segments. After deparaffinization and rehydration, antigens were recovered with 20 μg/mL proteinase K for 15 min at room temperature. Endogenous peroxidase was blocked with 3% H₂O₂ for 10 min to reduce nonspecific binding. After washing, sections were incubated in a humidified chamber at 37 °C for 1 h with TdT buffer containing TdT enzyme and reaction buffer. Specimens were incubated for 10 min at room temperature with a stop/wash buffer and then incubated in a humidified chamber for 30 min with anti-digoxigenin peroxidase conjugate at room temperature. After a series of PBS washes, slides were covered with peroxidase substrate to develop color and then washed in three changes of dH₂O and counterstained in 0.5% methyl green for 10 min at room temperature. TUNEL-positive cells were counted from digital images of at least ten different areas of each section (from 4 specimens per group) at 1000x using ImageJ software (NIH, Bethesda, MD, USA).

The detection of apoptotic cells was performed using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay (ApopTag®, Merck KGaA, Darmstadt, Germany) for light microscopy as already described in previous studies (27 (link)) and by Loo et al. (48 (link)). Accurate cell numbers in TUNEL slides were determined by image thresholding using ImageJ software (v1.46r, NIH, USA). The channels for red, blue and green were separated for better detection of apoptotic cells. The red area in the blue channel was analyzed with a pre-determined threshold for all images. The ratio of the counts in the red area (i.e., structures connected over several pixels indicating apoptotic cell nuclei) to the total marked area (counts/cm²) was analyzed. Five individual visual fields of one histological slide of the gut of each mouse were randomly chosen and microscopically recorded in 10 × magnification as previously described (27 (link)).

Apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated d-UTP nick-end labeling (TUNEL) staining using digoxigenin-labeled UTP detected with an antibody to digoxigenin using an in-situ apoptosis detection kit (Apoptag; CHEMICON, Schwalbach am Taunus, Germany), as described previously [83 (link)]. The apoptotic index also represented the percentage of TUNEL-positive cells relative to the total number of hepatocytes (400× magnification, five fields in each rat).

TUNEL assay for apoptosis was conducted according to the manufacturer’s protocol (ApopTag; Chemicon). Frozen sections (10 μm) from samples were prepared. The sections were counterstained with methyl green. At least three embryos of each genotype were used for each analysis.