Converter pod tunel kits

TUNEL was used for in situ detection of apoptosis in the liver and pancreas. Tissues were paraffin dewaxed to water, washed in phosphate buffered saline (PBS), pH 7.4 twice for 5 min each time, then incubated in 3% hydrogen peroxide for 20 minutes. Samples were then PBS washed twice more and dried. Reaction solution (TUNEL kits, Roche Diagnostics GmbH, Germany) was added to each sample (50 µl), and the reactions were performed at 37°C for 60 min in the dark. After incubation, samples were again washed twice in PBS and 50 µl Converter-POD (TUNEL kits, Roche Diagnostics GmbH, Germany) was added to the sample and incubated at 37°C for 30 min. Samples were washed again in PBS and a DAB Horseradish Peroxidase Color Development Kit (Beyotime Institute of Biotechnology, Shanghai China) was used to stain the samples. Hematoxylin was used to stain the nuclei. Samples then underwent conventional dehydration and were transparently mounted with glycerol. Colored nuclei were read as positive cells. The specimens were analyzed using a computerized image analysis system (IPP6.0 software, Media Cybernetics Inc, USA), and the data were represented as the number of TUNEL-positive cells per field (at 400X).

For in situ detection of apoptosis in the liver, parraffin wax sections were dewaxed to water and washed in 0.01 M PBS (pH 7) twice for 5 min, flooded with 3% hydrogen peroxide for 20 minutes, and finally washed with PBS twice for 5 min and dried. Fifty μl of reaction solution (TUNEL kits, Roche Diagnostics GmbH, Germany) was added to each sample, which was then incubated at 37°C for 60 min in the dark under humid conditions. The samples were then washed twice for 5 min in PBS. Fifty μl Converter-POD (TUNEL kits, Roche Diagnostics GmbH, Germany) was added to each sample, which was incubated at 37°C for 30 min and washed twice in PBS and stained with DAB Horseradish Peroxidase (DAB Horseradish Peroxidase Color Development Kit, Beyotime Institute of Biotechnology, Shanghai China). Finally, the samples were hematoxylin stained and underwent conventional dehydration and were transparently mounted with glycerol. In the subsequent images, nuclear staining was considered positive. The specimens were analyzed using a computerized image analysis system (IPP6.0 software, Media Cybernetics Inc, USA), and the percent of positive cells was calculated as followed: number of TUNEL positive cells / number of total hepatocytes counted. (at 400X)