HUVECs seeded in 24-well plates were fixed with 4% paraformaldehyde for 15 min after removing the culture medium. After fixation, the cells were washed three times with phosphate-buffered saline (PBS), then PBS containing 0.3% Triton X-100 was added, and the cells were incubated for 5 min at RT to remove the PBS. Next, 50 µl of terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling (TUNEL) assay solution (Beyotime, Shanghai, China) was added, and the cells were incubated at 37°C for 60 min in the dark and washed three times with PBS. The cells were sealed with an anti-fluorescence quenching liquid and observed under a fluorescence microscope.
Tunel assay solution
Manufactured by Beyotime
Sourced in China
The TUNEL assay solution is a reagent used in a laboratory technique that detects and quantifies apoptosis, a form of programmed cell death. The solution contains the necessary components to perform the Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, which labels the fragmented DNA in apoptotic cells.
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Lab products found in correlation
Triton x 100, by Beyotime (2 mentions)
4 6 diamidino 2 phenylindole dapi staining solution, by Beyotime (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Dapi staining, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Phosphate buffered saline (pbs), by Procell (1 mentions)
Dmi3000b, by Leica (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
9 protocols using tunel assay solution
1
TUNEL Assay for HUVECs Apoptosis
2
TUNEL Apoptosis Quantification in Brain Tissue
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Brain tissue was fixed with 4% PFA and embedded sections were cut. Dewaxing and protease addition were performed, followed by PBS washing. Each sample was then incubated dropwise with 50 μL of TUNEL assay solution (Beyotime, Shanghai, China) at 37°C for 60 minutes, and rewashed using PBS. Next, 4′,6-diamidino-2-phenylindole (DAPI) staining solution (Beyotime) was added to re-stain the nuclei. After washing, an antifade mounting medium was used. Apoptosis was observed and photographed using an ECHO revolve FL front inverted integrated fluorescence microscope (Cycloud, Beijing, China). Apoptosis rate (%) = TUNEL-positive cells/DAPI-positive cells × 100.
3
TUNEL Assay for Intestinal Tissue
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TUNEL staining of the intestinal tissue sections was performed. After dewaxing to water, the section was added with 20 ug/mL of proteinase K (positive control group was added with protease K containing 10 ug/mL DNase) at 37°C for 20 minutes. After that, the section was added with an appropriate amount of TUNEL assay solution (Beyotime, Hunan, China), and incubated at 37°C for 60 minutes in the dark. The section was washed 3 times with phosphate buffered saline (PBS) for 10 minutes each time, air-dried and sealed with anti-fluorescence quencher sealer.
4
Apoptosis Quantification in Cardiac Tissue
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The paraffin sections of the prepared cardiac tissues were placed in an oven at 60°C and baked for 2 h. After 20 μg/mL proteinase K was added, the sections were incubated at room temperature for 15 min. After rinsing with PBS, 50 μL of TUNEL assay solution (Beyotime, Shanghai, China) was added to the sections, which were incubated at 37°C for 1 h. The paraffin sections were stained with fluorescence dyes and observed under a fluorescence microscope (Nikon, Japan). Apoptotic cells were counted using Image-Pro Plus 6.0 image analysis software. The apoptosis rates of the cardiomyocytes were determined using the formula apoptotic index = number of apoptotic cardiomyocytes/the total number of cardiomyocytes × 100.
5
Apoptosis Detection in Cells
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AFCs were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min. Tunel assay solution (C1098, Beyotime) was prepared according to the reagent manufacturer’s instructions and incubated for 60 min in the dark. The nuclei were then stained with Hoechst 33,258. The samples were observed under the inverted fluorescence microscope (IX73, Olympus, Tokyo, Japan).
6
Gastrodin's Effects on OGD/R-Induced Apoptosis
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The effects of gastrodin on the apoptosis of OGD/R-induced R28 cells were detected by TUNEL staining. In brief, cells (1x105 cells/well) were rinsed by PBS for three times, followed by the fixation with 4% paraformaldehyde at room temperature. Subsequently, cells were probed with a small amount of DAPI staining (Beyotime, Cat. No. C1005) solution (covering the cells) and cultivated for 3–5 min at room temperature. 0.3% Triton-X-100 was also put into wells for further cultivation of cells. Afterward, 50 μl TUNEL assay solution (Beyotime, Cat. No. C1086) was employed to incubate the cells at 37°C in the dark for 60 min. Three fields of view were selected at random, and then cells were sealed with anti-fluorescence quenched sealing solution for observation under a fluorescence microscope (Zeiss GmbH, x200) [23 (link)].
7
TUNEL Assay for Apoptosis Detection
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Cell and testis sample treatment is described above. Cells were fixed with 4% PFA for 30 min and washed three times with phosphate-buffered saline (PBS). We added 0.3% Triton X-100 (Beyotime, China) to penetrate the cell membrane for 10 min, then washed the cells twice using PBS. The samples were added with 50 μL TUNEL assay solution (Beyotime, China) and incubated at 37 °C for 60 min, while protected from light. The cells were incubated with 1 µg/mL Hoechst 33342 dye solution at room temperature for 5 min in the dark, then washed with PBS. The samples with apoptotic cells were visualized by fluorescence microscopy.
Testis sections were subjected to the following reagents in sequence: xylene (1) for 10 min, xylene (2) for 10 min, absolute ethanol for 3 min, 90% alcohol for 3 min, 70% alcohol for 3 min, and distilled water for 2 min. We added 20 μg/mL DNase-free proteinase K to the sections, which were then incubated at 25 °C for 20 min and washed three times with PBS. The slides were incubated with terminal deoxynucleotidyl transferase (TdT) enzyme for 1 h at 37 °C, followed by counterstaining with Hoechst 33342 for 5 min. Finally, the sections were observed by fluorescence microscopy.
Testis sections were subjected to the following reagents in sequence: xylene (1) for 10 min, xylene (2) for 10 min, absolute ethanol for 3 min, 90% alcohol for 3 min, 70% alcohol for 3 min, and distilled water for 2 min. We added 20 μg/mL DNase-free proteinase K to the sections, which were then incubated at 25 °C for 20 min and washed three times with PBS. The slides were incubated with terminal deoxynucleotidyl transferase (TdT) enzyme for 1 h at 37 °C, followed by counterstaining with Hoechst 33342 for 5 min. Finally, the sections were observed by fluorescence microscopy.
8
TUNEL Assay for Apoptosis Detection
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After washing the cells once with PBS (Procell, Wuhan, China), the cells were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 30 min and then washed once more with PBS. PBS with 0.1% Triton X-100 (Beyotime, Shanghai, China) was added and incubated for 2 min on ice. 50 μl of the ready-made TUNEL assay solution (Beyotime, Shanghai, China) was added and incubated at 37° C in the dark for 60 min before washing three times with PBS. TUNEL-positive cells were observed under fluorescence microscope after being treated with anti-fluorescence quenching solution.
9
Apoptosis Assessment in Mouse Myocardial Cells
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TUNEL assay was performed to assess the apoptosis of myocardial cells in reperfused mouse hearts. Paraffin-embedded tissues were sectioned, dewaxed, dehydrated, and mounted on slides. The tissue sections were treated with proteinase K at 37°C for 30 min. After washing with phosphate-buffered saline (pH 7.4) solution thrice, 50 μL of TUNEL assay solution (#C1091; Beyotime, Shanghai, China) was added, and the slides were incubated at 37°C for 30 min in the dark, sealed with ProLong™ Gold Antifade Mountant with DAPI (#P36931; Thermo Fisher Scientific, Waltham, MA, United States), and observed under a fluorescence microscope (DMI3000 B, Leica Microsystems).
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