The left ventricles were fixed overnight in 4% paraformaldehyde, embedded in paraffin, and then cut into 5-μm sections. After staining with TdT-Mediated dUTP Nick-End Labeling (TUNEL) (Beyotime, Shanghai, China) at 37°C for 60 min, the sections were washed three times with PBS, then observed, and photographed under an optical microscope. The quantification was performed using ImageJ software.
Tdt mediated dutp nick end labeling tunel
Manufactured by Beyotime
Sourced in China
The TdT-Mediated dUTP Nick-End Labeling (TUNEL) is a method for detecting and quantifying apoptosis, or programmed cell death, in cells. The technique uses terminal deoxynucleotidyl transferase (TdT) to label the free 3'-hydroxyl ends of DNA fragments generated during apoptosis. The labeled DNA can then be detected and quantified using various techniques, such as fluorescence microscopy or flow cytometry.
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3 protocols using tdt mediated dutp nick end labeling tunel
1
Quantifying Cardiomyocyte Apoptosis
2
Quantifying Endothelial Cell Apoptosis
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DNA fragmentation of endothelial cells was measured by TdT-mediated dUTP nick-end labeling (TUNEL) staining in line with the manufacturer’s protocol (Beyotime). In brief, endothelial cells were fixed and permeabilized, as described earlier. After rinsing with PBS, the cells were incubated with a one-step TUNEL reaction mixture for 1 h at 37°C in a humidified chamber in the dark. Finally, nuclei were counterstained with DAPI (Beyotime). Images were observed, and photographs were taken under a fluorescence microscope (Olympus).
3
Apoptosis Assessment by TUNEL and Annexin V-FITC/PI
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TdT-mediated dUTP Nick-End Labeling (TUNEL) (Beyotime, China) and Annexin V-fluorescein isothiocyanate (AV-FITC)/propidium iodide (PI) (BD Biosciences, San Jose, CA, USA) were used to evaluate cell apoptosis. Among these assays, the TUNEL positive cells to total cells ratio was calculated to assess the level of apoptosis, and the Annexin V-FITC/PI assay was performed to analyze the percentage of early apoptotic as well as late apoptotic cells. The above assays were performed by referring to the manufacturers’ instructions and our previous studies.22 (link),24 (link)
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