The lung sections (5 μM) were stained via terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining (Beyotime Biotech Inc., Nantong, China) to examine the apoptosis of cells in mouse lung according to the manufacturer’s instructions. Murine lung sections without any stimuli were incubated with 0.01 U/ul DNase I for 10 min at room temperature (RT) and were regarded as a positive control. For quantification of apoptotic cells, 20 random fields per section in each group were counted, and the average number of apoptosis per section was calculated.
Tunel staining
Manufactured by Beyotime
Sourced in China
TUNEL staining is a technique used to detect and visualize DNA fragmentation, a hallmark of apoptosis or programmed cell death. The TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay incorporates modified nucleotides into the free 3'-hydroxyl ends of DNA fragments, allowing for the identification of cells undergoing apoptosis.
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Lab products found in correlation
Image pro plus 6, by Media Cybernetics (2 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
Invitrogen evos fl auto, by Thermo Fisher Scientific (1 mentions)
Confocal microscopy, by Zeiss (1 mentions)
Em uc7 microtome, by Leica (1 mentions)
Axioplan 2 imaging system, by Zeiss (1 mentions)
Annexin 5 fitc staining, by Beyotime (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
Rapamycin rap, by Selleck Chemicals (1 mentions)
Standard msc expansion medium, by Cyagen (1 mentions)
Primary antibodies against beclin 1, by Cell Signaling Technology (1 mentions)
Cobalt protoporphyrin 9 copp, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
β actin, by Proteintech (1 mentions)
Ros detection kit, by Beyotime (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Beyotime (1 mentions)
Hydrogen peroxide h2o2, by Sangon (1 mentions)
Fluorescein isothiocyanate (fitc), by Beyotime (1 mentions)
Bright field microscopy, by Nikon (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
30 protocols using tunel staining
1
TUNEL Staining of Apoptotic Cells in Mouse Lung
2
TUNEL Staining for Apoptosis Analysis
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TUNEL staining (Beyotime Institute of Biotechnology) was used to analyze cell apoptosis. Following fixation with 4% paraformaldehyde for 24 h at room temperature, sections of murine colons were rinsed in distilled water and incubated with 3% hydrogen peroxide in methanol for 5 min at room temperature to block endogenous peroxidase activity, following which the samples were deparaffinized using xylene and rehydrated in a descending ethanol series. The sections were incubated with 20 µg/ml proteinase K (Dako; Agilent Technologies, Inc.) for 15 min at room temperature, and TdT enzyme solution was added and incubated for 1 h at 37°C. The sections were then incubated with streptavidin-peroxidase conjugate for 30 min at 37°C. Peroxidase activity was demonstrated by the addition of 50 µl DAB for 10 min at room temperature and a light microscope was used to observe apoptosis in six randomly selected fields of view (magnification, ×200).
3
Apoptosis Detection in Tumor Tissue Samples
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TUNEL staining was used to detect apoptosis in the resected tissue samples. Frozen tumor tissue sections (3–5 µm; stored at −20°C) from the indicated nude mice were processed for TUNEL staining (Beyotime Institute of Biotechnology), according to the manufacturer's instructions. Briefly, following fixation by 4% paraformaldehyde for 20–30 min at room temperature, the frozen sections were washed three times with PBS at room temperature (5 min each time). Then, sections were immersed in PBS containing 1% Triton X-100 for 20 min at room temperature. Each sample was incubated with 50 µl TdT enzyme reaction solution (45 µl equilibration buffer, 1 µl biotin-11-dUTP and 4 µl TdT enzyme) for 60 min at RT. Following PBS washing, 50 µl streptavidin-TRITC labeling buffer was added and incubated for 30 min at room temperature. Then, sections were covered with DAPI (1:1,000) for 10 min at RT. Finally, the sections were placed under a cover slip and scanned by Invitrogen EVOS FL AUTO (Thermo Fisher Scientific, Inc.).
4
Evaluating SENP3 Modulation of HAEC Apoptosis
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HAECs at a density of 30,000 cells/well were seeded into 24-well plates, followed by SENP3 lentivirus infection and high glucose treatment for 24 h. Cell apoptosis was assayed by TUNEL staining according to the manufacturer’s instructions (Beyotime, Nantong, China).
5
Cardiomyocyte Apoptosis Analysis
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Two hours after reperfusion, the myocardium of the ischemic area was dissected and fixed in 4% paraformaldehyde at 4 °C overnight, and then embedded in paraffin. TUNEL staining was performed as described (Beyotime Biotechnology Co., Ltd., Shanghai) [22 (link)]. Cardiomyocyte apoptosis in the myocardium was observed by confocal microscopy (Carl Zeiss, Germany).
6
Quantification of Brain Infarct and Apoptosis
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The SD rats were sacrificed at 7 days after theaflavin treatment. The harvested brain tissues were cut into five equally-sized coronal sections (2–3 mm thickness) using an EM UC7 microtome (Leica Microsystems Inc., Wetzlar, Germany). The brain sections were then incubated with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich; Merck KGaA) at 37°C for 15 min with gentle shaking. Subsequently, the brain sections were fixed with paraformaldehyde (4%) overnight. The stained slices were photographed and the infarct size was quantified using the Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (Beyotime Institute of Biotechnology, Haimen, China), the harvested brain tissues were cut into 2.5 mm blocks and fixed with paraformaldehyde overnight. Subsequently, blocks were embedded in paraffin and cut into 4-µm coronal sections, according to the manufacturer's protocol. Apoptotic images were obtained using the Axioplan 2 imaging system (Carl Zeiss AG, Oberkochen, Germany). Six sections of each sample were selected and five fields of each section were examined for quantification of apoptosis.
7
Apoptosis Analysis by TUNEL and Annexin V
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Apoptosis was analyzed by TUNEL assay, and flow cytometry. TUNEL staining (Beyotime Biotechnology, China) was performed following the manufacturer’s instructions. For Annexin V-FITC staining (Beyotime Biotechnology, China), the cells were suspended in 100 μL of Binding Buffer and then were treated with 5 μL Annexin V-FITC and 5 μL of PI in the dark for 15 min. Finally, they were analyzed by flow cytometry.
8
Fixation and TUNEL Staining Protocol
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The cells were fixed with 4% PFA at RT for 30 min, then TUNEL staining (Beyotime) was used according to manufacturer's instructions.
9
APAP-Induced Liver Injury in Mice
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APAP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was dissolved in phosphate-buffered saline. Mice (8 weeks old) were fasted overnight and injected with APAP intraperitoneally at the dose of 400 mg/kg to induce hepatotoxicity and at the dose of 600 mg/kg to monitor survival rate. The serum was collected for alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) assay. The tissue homogenates from the mouse liver were used to evaluate the levels of GSH with a commercial kit (Jiancheng Biotech, Nanjing, China). Liver damage was detected by hematoxylin and eosin (H&E) staining. Cell death was evaluated by TUNEL staining (Beyotime Biotechnology, Shanghai, China) according to the manufacturer's instruction. In some cases, male WT and fat-1 mice were injected with 100 mg/kg β-estradiol (E2) intraperitoneally 1 week before APAP administration. To test the effect of exogenous n-3 PUFAs on APAP toxicity, male and female WT mice were fed with an n-3 PUFA-enriched diet 3 weeks before APAP injection.
10
Hydrogen Peroxide Modulation of Mesenchymal Stem Cells
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Hydrogen peroxide (H2O2) was purchased from Sangon Biotech (Shanghai, China). Cobalt protoporphyrin IX (CoPP) was purchased from Sigma (USA). Rapamycin (RAP) was purchased from Selleck (USA). The standard MSC expansion medium was purchased from Cyagen Biosciences Inc. (Guangzhou, China). Cell counting kit-8 (CCK-8) was acquired from Dojindo (Japan). ROS detection kit, TUNEL staining, Annexin V-FITC/PI Apoptosis Detection Kit, and Quick Block™ Blocking Buffer were purchased from Beyotime Institute of Biotechnology (Beyotime, China). The rat heme oxygenase-1- (HO-1-) siRNAs were designed and manufactured by Ribobio Co., Ltd. (Guangzhou, China). The mRFP-GFP-LC3 adenovirus was purchased from HanBio Technology Co., Ltd. (Shanghai, China). Primary antibodies against HO-1, β-actin, and the secondary antibodies were purchased from Proteintech (Wuhan, China). Primary antibodies against LC3-I/II were purchased from Abcam (UK). Primary antibodies against Beclin-1 were purchased from Cell Signaling Technology (USA).
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