Tunel pod

The free 3’-OH groups in broken cellular DNA resulting from apoptotic degradation were detected using the TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling), which was performed according to the manufacturer’s instructions. Pancreatic tissue sections (4 μm) were mounted on a slide, dewaxed and rehydrated in graded alcohol solutions. Immunohistochemical detection and quantification of terminal transferase dUTP nick end labeling (TUNEL) were performed using an in situ cell death detection kit (TUNEL-POD; Roche, Basel, Switzerland) following the manufacturer’s instructions. Apoptotic cells (brown staining) were visualized using an Olympus microscope under 400x magnification. The apoptotic index (AI) was expressed as the means ± S.D. for the percentage of positive cells and was calculated using the formula:
$AI=(apoptosis-positivecells⁄totalcells)\times 100\%.$
Two slides were selected from each animal, and five fields (400x) in each slide were randomly selected to quantify the positive cell number per 1,000 cells.

Dewaxed sections were rehydrated, and antigen retrieval achieved by incubating the sections with 15 μg/ml Proteinase K in 10 mM Tris-HCL at pH 7.5 (03115887001, Roche, Basel, Switzerland) for 30 min in a humidified chamber. The sections were washed three times in PBS for 10 min each, incubated with freshly prepared TUNEL reaction mixture (TUNEL Enzyme solution diluted 1:12.25 in TUNEL Label solution, (11684817910, Roche, Basel, Switzerland)) for 60 min at 37°C in a humidified incubator. Subsequently sections were incubated with anti-fluorescein antibody conjugated to horseradish peroxidise, TUNEL-POD (1772465, Roche, Basel, Switzerland) diluted 1:2 in PBS, for 30 min at 37°C in a humidified incubator. The sections were washed three times in PBS for 10 min each, incubated with DAB chromogen-substrate buffer solution (K3468, DAKO, Baar, Switzerland) for 5–10 min until a brown signal was detected and immersed in PBS to stop the reaction. The sections were washed for 2 min under running distilled water and counterstaining as above

Survivin protein expression levels in the craniopharyngioma tissues were detected using immunohistochemistry and then quantitatively analyzed. Apoptotic cells were stained with transferase dUTP nick end labeling-peroxidase (TUNEL-POD; Roche, Branchburg, NJ, USA) and observed using transmission electron microscopy (Hitachi H-600IV; Hitachi, Ltd., Tokyo, Japan). Under the light microscope, survivin-positive cells were observed as brown granules in the nucleus or cytoplasm. For each section, a charge-coupled device camera was used to capture at least five fields of view at a magnification of ×400 (Olympus BX51 CCD; P70; Olympus Corporation, Tokyo, Japan), and Image-Pro Plus software (version 5.0; Media Cybernetics, Rockville, MD, USA) was used to quantitatively analyze the images; integrated optical density (IOD) was selected as the parameter to indicate survivin protein expression levels and the average IOD of all the recorded images was determined. Furthermore, TUNEL-POD stained the nuclei of the apoptotic cells yellow or brown, while the nuclei of the non-apoptotic cells were blue.