IPEC-J2 cells were grown on microscope cover slips placed in 6-well tissue culture plates and mock-infected or infected with PEDV at an MOI of 1.0.The virus-infected cells were fixed at 48 hpi with 4% paraformaldehyde for 25 min at 4 °C and permeabilized with 0.2% TritonX-100 in PBS at room temperature for 5 min. Cell samples were rinsed twice with PBS, and the TUNEL reaction mixture (Beyotime Biotechnology) was added and incubated for 60 min at 37 °C, followed by three washes with PBS. TUNEL-labeled cells were subjected to immunofluorescence assay using anti-PEDV N protein mouse monoclonal antibody and goat anti-mouse antibody, as described above. After sealing with an anti-fluorescence quenching liquid, the samples were mounted on a fluorescence microscope and examined at an excitation wavelength of 550 nm and emission wavelength of 570 nm (red fluorescence).
Tunel reaction mixture
Manufactured by Beyotime
Sourced in China
The TUNEL reaction mixture is a reagent used in the Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assay. The TUNEL assay is a method for detecting and quantifying apoptosis, or programmed cell death, in cells. The TUNEL reaction mixture contains the necessary components, including terminal deoxynucleotidyl transferase (TdT) enzyme and labeled nucleotides, to detect and label the DNA strand breaks that are characteristic of apoptotic cells.
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Lab products found in correlation
Fluorescence microscope, by Olympus (2 mentions)
Ab177487, by Abcam (1 mentions)
Fluorescence microscope, by Motic (1 mentions)
Ab25939, by Abcam (1 mentions)
Ab6939, by Abcam (1 mentions)
Inverted fluorescent microscope, by Leica camera (1 mentions)
St532, by Beyotime (1 mentions)
F8775, by Merck Group (1 mentions)
Leica dm3000 microscope, by Leica camera (1 mentions)
Flow cytometry, by BD (1 mentions)
Hoechst 33358, by Merck Group (1 mentions)
Laser scanning biological microscope fv1000, by Olympus (1 mentions)
Confocal dishes, by NEST Biotechnology (1 mentions)
Antifade mounting medium, by Beyotime (1 mentions)
Alexa fluor plus 594, by Thermo Fisher Scientific (1 mentions)
Dnase free protease k, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Goat serum, by Beyotime (1 mentions)
Leica confocal microscope, by Leica Microsystems (1 mentions)
Dab kit, by Beyotime (1 mentions)
27 protocols using tunel reaction mixture
1
PEDV Infection and TUNEL Assay
2
Apoptosis assessment in rat cortex
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Apoptosis of neurons in the ipsilateral cortex of SD rats was evaluated by terminal-deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) and NeuN double immunostaining according to the manufacturer’s protocol. The brain sections from each group were incubated with TUNEL reaction mixture (Beyotime, Shanghai, China) for 1 h at room temperature and then stained with anti-NeuN (ab177487, Abcam, Cambridge, United Kingdom) and DAPI (Wellbio, China). The slides were observed using a fluorescence microscope (Motic, China).
3
Dual Immunofluorescence Analysis of Brain Tissue
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For terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and neuronal nuclear protein (NeuN) double staining, paraffin sections of the brain tissue on the 3rd, 7th, and 14th day after modeling were dewaxed and rehydrated by xylene and graded alcohol and then incubated at 37°C in ethylenediaminetetraacetic acid (EDTA) (pH 9.0) for 15 minutes. A TUNEL reaction mixture (Beyotime, China) was added and then blocked with 3% goat serum albumin for 30 min. The sections were then incubated with primary antibody against NeuN (0.72 μg/ml, 24307, CST) at 4°C overnight and Cy3-labeled goat anti-rabbit IgG antibody (0.8 μg/ml, ab6939, Abcam) for 50 min sequentially.
For cIAP1 and NeuN double staining, paraffin sections of the brain tissue on the 3rd, 7th, and 14th day after modeling were dewaxed and rehydrated by xylene and graded alcohol and then incubated in 37°C EDTA (pH 9.0) for 15 minutes. The sections were then incubated with primary antibody against NeuN (0.72 μg/ml, 24307, CST) and cIAP1 (5 μg/ml, ab25939, Abcam) at 4°C overnight. Then, Cy3-labeled goat anti-rabbit IgG antibody (0.8 μg/ml, ab6939, Abcam) was added and incubated for 50 minutes sequentially. Finally, the sections were incubated with DAPI for 10 min prior to visualizing them with a fluorescence microscope (BX51, Olympus, Japan) at 400 magnification.
For cIAP1 and NeuN double staining, paraffin sections of the brain tissue on the 3rd, 7th, and 14th day after modeling were dewaxed and rehydrated by xylene and graded alcohol and then incubated in 37°C EDTA (pH 9.0) for 15 minutes. The sections were then incubated with primary antibody against NeuN (0.72 μg/ml, 24307, CST) and cIAP1 (5 μg/ml, ab25939, Abcam) at 4°C overnight. Then, Cy3-labeled goat anti-rabbit IgG antibody (0.8 μg/ml, ab6939, Abcam) was added and incubated for 50 minutes sequentially. Finally, the sections were incubated with DAPI for 10 min prior to visualizing them with a fluorescence microscope (BX51, Olympus, Japan) at 400 magnification.
4
Neuroprotective Effect of PNU-282987
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To observe the neuroprotective effect of PNU-282987, TUNEL assays were used to detect apoptotic cells. After deparaffinization in dimethylbenzene, the brain tissue sections were rehydrated in gradient ethanol (concentrations ranging from 100% to 70%), washed with pure water and PBS, incubated with proteinase K for 10 min before incubation in the TUNEL reaction mixture (beyotime, China) for 1 hour in the dark at 37°C. Finally, the sections were added with antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI). With an inverted fluorescent microscope (Leica, Germany), TUNEL positive cells were observed at 520 nm of green fluorescence, nuclei were observed at 460 nm of blue fluorescence. Image J software was used to calculate the number of apoptotic cells in the hippocampus.
5
TUNEL Staining for Tumor Apoptosis
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In order to detect tumor apoptosis, TUNEL staining was performed. The fixed tumor tissues were embedded in paraffin and 5-μm-thick sections were deparaffinized by washing in xylene and a descending ethanol series. The sections were subsequently incubated with 20 μg/mL proteinase K for 30 min at 37 °C, and endogenous peroxidase was inactivated by 3 % H2O2 in methanol for 10 min. They were incubated with 50 μL of a TUNEL reaction mixture (Beyotime Biotechnology) on the section for 60 min at 37 °C, and then 50 μL converter-POD was added to the sample for 30 min at 37 °C. For color development, sections were supplement-ed with 50 μL DAB substrate for 10 min at room temperature to detect labeled nuclei, and then counterstained by hematoxylin. For each slide, 5∼10 separate fields were examined randomly and digitized by microscopy. The apoptotic index was calculated as the ratio of TUNEL-positive cells to the total number of cells.
6
TUNEL Assay for Apoptosis Detection
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The sample was fixed in formalin (F8775, Sigma-Aldrich, Shanghai, China) and cut into 3 μm slice after paraffin embedding. Tissue sections were pre-treated proteinase K working solution (20 μg/mL in 10 mM Tris/HCl, pH 7.4-8, ST532, Beyotime, Shanghai, China) for 15 min at room temperature after the tissue sections were dewaxed and rehydrated. PBS rinse the slides 3 times. Add 50 μL of TUNEL reaction mixture (C1088, Beyotime, Shanghai, China) to the sample and incubate for 60 min in a dark and humidified chamber at 37 °C. After three times PBS rinse, DAPI (D212, 1:5000, Dojindo, Beijing, China) was used to stain the nuclei. The immunofluorescent images were captured under a fluorescence microscope (Olympus, Tokyo, Japan). The image was then quantified and analyzed by Image J (version 1.8.0).
7
Histological and Apoptosis Analysis of Ovarian Tissues
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Then, ovarian tissues were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (H&E). Then, sections were viewed and photographed using a Leica DM3000 microscope (Leica, Germany). TUNEL staining was performed to assess the cell apoptosis in ovarian tissues. Briefly, ovarian tissue sections were incubated with the TUNEL reaction mixture in a humidified chamber at 37°C for 1 h (Beyotime Biotechnology Co., Ltd., Shanghai) and further incubated with peroxidase-conjugated HRP antibody at 37°C for 30 min. Then, immunoreactivity was visualized using 3,3′-diaminobenzidine (DBA), and the TUNEL-positive cells in ovarian tissue sections were observed and photographed using a Leica DM3000 microscope (Leica, Germany).
8
Quantifying Apoptosis in Cells and Tissue
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Apoptosis in cultured cells was determined by flow cytometry and Hoechst 33258 staining. For the flow cytometry assay, the harvested cells were fixed with pre-cooled 70% ethanol at 4°C overnight; after which, they were washed twice with PBS and then suspended in Annexin-V Binding Buffer at a density of 2–3 × 106 cells/ml. Next, Annexin-V FITC and propidium iodide (PI) buffers were added to the cell suspension and the mixture was incubated at room temperature for 15 min while being protected from light. The cells were then analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, U.S.A.). For the Hoechst 33358 (Sigma, U.S.A.) staining assay, the cultured cells were fixed with 4% PFA at room temperature for 15 min, and then incubated in the dark with Hoechst 33258 (10 μg/ml) solution for 5 min. The stained cells were then observed with a fluorescence microscope.
Cell apoptosis in samples of lung tissue was examined by the Terminal Transferase dUTP Nick End Labeling (TUNEL) assay. In brief, 5 μm-thick sections of lung tissue fixed in neutral formalin were prepared as described above and then incubated with a TUNEL reaction mixture (Beyotime, Shanghai, China) for 1 h at room temperature in a humidified incubator. TUNEL-positive cells, which displayed a brown color, were analyzed under a fluorescence microscope.
Cell apoptosis in samples of lung tissue was examined by the Terminal Transferase dUTP Nick End Labeling (TUNEL) assay. In brief, 5 μm-thick sections of lung tissue fixed in neutral formalin were prepared as described above and then incubated with a TUNEL reaction mixture (Beyotime, Shanghai, China) for 1 h at room temperature in a humidified incubator. TUNEL-positive cells, which displayed a brown color, were analyzed under a fluorescence microscope.
9
Quantifying Cardiomyocyte Apoptosis via TUNEL
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Treated H9c2 cells cultured in 6-well plates were fixed with 4% neutral formaldehyde for 20 min at 4°C. After washing with PBS, cells were permeabilized on ice with 0.1% Triton X-100 in PBS for 2 min. Washed cells were incubated for 60 min with 50 µl/well TUNEL reaction mixture (Beyotime, Shanghai, China), which containing nucleotide mixture and terminal deoxynucleotidyl transferase. At last, the cells were then washed by PBS for 3 times and observed under fluorescence microscope (Olympus BX43, Tokyo, Japan). TUNEL-positive cells were random counted in 5 randomly fields per section, then quantified by image analysis system. The apoptosis rate was expressed as the ratio of TUNEL-positive cardiomyocytes number to total number of cardiomyocytes.
10
Apoptosis Identification in Hippocampus
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Sections of hippocampal tissues were incubated with proteinase K solution for 15 min followed by incubation with TUNEL reaction mixture (Beyotime). After washing three times with PBS, the sections were mounted with a mounting solution containing the nuclear dye DAPI and observed using either a conventional fluorescence microscope or a confocal laser fluorescence microscope.
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