In order to detect apoptosis, TUNEL assay was used as previously described25 (link). Briefly, cells were fixed with 4% paraformaldehyde solution for 0.5 h at room temperature. Then the cells were exposed to a methanol solution containing 0.2% H2O2 for 0.5 h to block endogenous peroxidase activity. TUNEL reaction mixture (Sigma-Aldrich) was then added, and the cells were placed in an incubator at 37°C for 60 min. Finally, laser confocal microscopy (A1; Nikon, Japan) was used to capture the fluorescence. Apoptosis index was quantified as the ratio of (TUNEL+ cells)/(total cells) × 100%.
Tunel reaction mixture
Manufactured by Merck Group
Sourced in United States
The TUNEL reaction mixture is a laboratory reagent used to detect and visualize DNA fragmentation associated with apoptosis, or programmed cell death. The core function of this product is to provide the necessary components for the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay, a commonly used technique for the detection of apoptotic cells.
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Lab products found in correlation
Simvastatin, by Merck Group (1 mentions)
Tunel fluorescence fitc kit, by Roche (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
3 3 diaminobenzidine (dab), by Beyotime (1 mentions)
8 well culture slides, by Corning (1 mentions)
Fluorescence microscopy, by Leica (1 mentions)
Mmp13, by Proteintech (1 mentions)
Ti2 e, by Nikon (1 mentions)
Fast green, by Merck Group (1 mentions)
Alexa fluor 555 conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Fast green solution, by Merck Group (1 mentions)
Safranin o solution, by Merck Group (1 mentions)
Rabbit anti nanog, by Abcam (1 mentions)
Goat anti sox17, by R&D Systems (1 mentions)
Mouse anti cdx2, by BioGenex (1 mentions)
Fv1000, by Leica (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Polyvinylpyrrolidone, by Merck Group (1 mentions)
Tunel brightred apoptosis detection kit, by Vazyme (1 mentions)
13 protocols using tunel reaction mixture
1
Quantifying Apoptosis via TUNEL Assay
2
Quantifying Apoptosis in Myocardial Tissue
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The TUNEL assay was used for the analysis of apoptosis of myocardial tissue in experimental mice at 37°C following simvastatin treatment (10 mg/kg/day; Sigma-Aldrich; Merck KGaA) or an equivalent dose of PBS. Procedures were performed as previously described (27 (link)). Briefly, 4-µm tissue sections were fixed with 4% paraformaldehyde at 37°C followed by permeabilization with 0.1% Triton X-100 for 1 h at 37°C. Subsequently, tissue sections or myocardial cells (1×106) were stained with TUNEL reaction mixture (Sigma-Aldrich; Merck KGaA) at 37°C for 2 h. Tissue sections and myocardial cells were washed 3 times in TBS-Tween-20. TUNEL assays were conducted using a TUNEL fluorescence FITC kit (Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. Samples were mounted with neutral gum. Images in 6 fields of view (magnification, ×400) were captured using a Zeiss LSM 510 confocal microscope (Zeiss AG, Oberkochen, Germany) at a wavelength of 488 nm.
3
Retina Cell Death Apoptosis Assay
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We investigated cell death in the retina as previously described [30 (link)]. We performed Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay for the detection of apoptosis in situ. The nucleotide-labeling mix was used in combination with the TUNEL enzyme to prepare a TUNEL reaction mixture (all from Sigma-Aldrich, city, State, USA) and the assay was performed according to the manufacturer’s instructions. The number of TUNEL-positive cells was counted in 3–5 cross-sections of the retina per animal. Results are expressed as the average number of TUNEL-positive cells per section ± SEM.
4
Apoptosis Detection in Brain Tissue
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The paraffin sections were permeabilized and treated with 50 μl TUNEL reaction mixture (Sigma Aldrich, St. Louis, MO, USA) for 60 min at 37°C with no light. Then, the slides were treated with 50 μl peroxidase (POD, Beyotime) for 30 min, followed by incubation with diaminobenzidine substrate solution (50 μl, DAB, Beyotime) for 10 min at 25°C. The TUNEL-positive apoptotic cells in the brain tissues were analyzed using an optical microscope (Tokyo, Japan).
5
Apoptosis Detection in Retina
6
Apoptosis Detection by TUNEL Assay
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To detect the apoptosis based on the detection of single- and double-stranded DNA breaks a tunel assay was carried out. SF-268 (3 × 104 cells) were seeded in 8 well culture slides (Corning, USA), exposed to active principles (IC50) for 48 h, fixed with 100 µL of paraformaldehyde (4% in PBS, pH 7.4) and incubated 60 min at 25 °C. Then, cells were washed with PBS and resuspended with 100 µL/well permeabilisation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice at 4 °C. Cells were washed again with PBS, resuspended in 50 µL/wll Tunel reaction mixture (Sigma Aldrich, Madrid, Spain) and incubated 1 h at 37 °C in a humidified atmosphere in the dark. Finally, cells were washed with PBS and observed by fluorescence microscopy (Leica Microsystems, Wetzlar, Germany).
7
Comprehensive Joint Tissue Analysis
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The knee joints from human and mice were fixed in 4% PFA (biosharp) and then decalcified with EDTA. For Safranin O-Fast Green staining, each paraffin-embedded sample was sectioned at 4 μm, and was stained with 0.2% Safranin O solution for 15 min and 0.2% Fast Green solution for 5 min (Sigma-Aldrich, USA). For immunofluorescence, after conventional dewaxing, the sections were repaired with EDTA repair solution in a boiling water bath for 30 min. Then, 0.5% Triton (Regan) was used to permeate the membrane for 20 min and 5% BSA (MCE, China) was used for blocking for 1 h. After that, the slices were incubated with primary antibodies (diluted 1:100 by BSA, MMP3: Abcam Cat# ab52915, RRID: AB_881,243; MMP13:Proteintech Cat# 18,165–1-AP, RRID: AB_2,144,858) at 4 °C overnight, washed thrice with PBST and incubated with the fluorescent Alexa Fluor® 555-conjugated secondary antibody (Cell Signaling Technology Cat# 4413, RRID: AB_10,694,110) at 37 °C for 1 h. Finally, the nuclei were stained with DAPI. Fluorescence images were acquired using Nikon Ti2-E. For TUNEL staining, after incubation with 0.5% Triton (Regan) for 15 min, rinsing slide(s) three times in PBS for 5 min each, adding 25 µL TUNEL reaction mixture (Sigma, Roche) on the section for 1 h at 37 °C in dark. Evaluated the section under a fluorescence microscope in the range of 523–555 nm (green).
8
Immunofluorescence Imaging of Embryos
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Embryos were treated with AT solution to remove the zona pellucida and fixed in 4% PFA for 20 min at room temperature. The embryos were then permeabilized for 20 min in 0.6% Triton X-100 (Sigma), washed in PBS and incubated in blocking solution (3% BSA (Sigma) in PBS) at 4 °C for 4 h and then with primary antibodies in blocking solution for a minimum of 6 h at 4 °C. They were then washed and incubated with secondary antibodies for 1 h, washed again and incubated with DAPI for 5 min. Primary antibodies used: mouse anti-Cdx2 (1:200; BioGenex), rabbit anti-Nanog (1:200; Abcam) and goat anti-Sox17 (1:200; R&D Systems). For TUNEL staining, the embryos were fixed, washed and permeabilized for 30 min in 0.5% Triton-X100 on ice. The embryos were then washed in PBS/PVP (PBS containing 1 mg ml−1 polyvinylpyrrolidone (Sigma) and incubated in 50 μl of TUNEL reaction mixture (Sigma) in the dark for 1 h at 37 °C. Embryos were then washed through PBS/PVP and incubated in DAPI for 5 min at room temperature before imaging. Confocal imaging was carried out using either an Olympus FV1000 (Fluoview FV10-AW software) or Leica SP5 (LAS AF software) inverted confocal microscope. Image files were viewed and analysed using ImageJ (http://imagej.nih.gov/ij/ ) or Fiji (www.fiji.sc/wiki/index.php/Fiji ) software.
9
TUNEL Assay for Apoptosis Detection
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Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. Apoptosis was evaluated with the TUNEL BrightRed Apoptosis Detection kit (Vazyme Biotech Co., Ltd. Nanjing, China) according to the manufacturer’s instructions. Briefly, 5 × 104 cells/ml were plated in 6-well flat-bottom plates and pretreated with 1 μM Rap for 1 h and then treated with 0.5 mM MG for 36 h. Cells were fixed in 4% paraformaldehyde at 4 °C for 30 min, and then permeabilized in 0.1% Triton X-100. Cells were then washed and stained with TUNEL reaction mixture and DAPI (Sigma, St. Louis, MO). The image was visualized and captured by microscope (Olympus × 51 W, Olympus Microsystems).
10
Quantifying Apoptosis in Tumor Samples
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Tumors were harvested and fixed with 10% neutral-buffered formalin. Deparaffinized sections were incubated with 20 μg/mL protease K for 15 min at room temperature, washed with PBS, and incubated with the TUNEL reaction mixture (Millipore, Burlington, MA, USA) for 1 h at 37 °C in a humidified chamber. The analysis was carried out in a blinded manner.
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