The tissues of all groups were examined using a Terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) kit (Beyotime, Beijing, China) to generate the apoptosis index. TUNEL staining was operated with a TUNEL kit (Beyotime) in accordance with the manufacturer's protocol. The tissue sections were floated in a water bath for 60 min at 37°C and washed five times in poly(butylene succinate‐eo‐terephthalate). The sections were incubated with corresponding antibodies in the dark at 37°C and added with peroxidase. The staining was visualized using 3, 3′‐diaminobenzidine. Then, the sections were sealed followed by 12 h of air drying. Under a light microscope with ×400 magnification in five fields of view, the sections of each group were observed and photographed. The number of positive retinal neurons and total retinal neurons were calculated. The apoptosis index calculation was carried out as follows: apoptosis index = the number of positive retinal neurons/the number of total retinal neurons.
Tunel kit
Manufactured by Beyotime
Sourced in China, United States
The TUNEL kit is a laboratory tool used for the detection and analysis of DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides a method to label the free 3'-hydroxyl termini in the fragmented DNA, allowing for the visualization and quantification of apoptotic cells.
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Lab products found in correlation
Fluorescence microscope, by Olympus (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (6 mentions)
Proteinase k, by Beyotime (4 mentions)
Fluorescence microscope, by Nikon (3 mentions)
Fluorescence microscope, by Leica camera (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Picric acid, by Solarbio (2 mentions)
β gal kit, by Beyotime (2 mentions)
Mayer s h e, by Nanjing Jiancheng (2 mentions)
Anti 53bp1, by Novus Biologicals (2 mentions)
Anti nephrin, by Abcam (2 mentions)
Zen2012, by Olympus (2 mentions)
Dnase free proteinase k, by Beyotime (2 mentions)
Evos fl microscope, by Thermo Fisher Scientific (2 mentions)
Lms710, by Zeiss (2 mentions)
Diaminobenzidine, by ZSGB-BIO (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Proteinase k, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Eclipse ni u microscope, by Nikon (2 mentions)
170 protocols using tunel kit
1
TUNEL Assay for Retinal Neuron Apoptosis
2
TUNEL Assay for Apoptosis Quantification
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The apoptosis of HT-22 cells and brain tissues from rats were detected by TUNEL assay using a TUNEL kit (Beyotime, Shanghai, China). Images were captured using a microscope (Olympus, Japan), and TUNEL-positive cells were counted using ImageJ.
3
Carbonyl Sulfide Assay and Cytokine Analysis
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Carbonyl sulfide was purchased from the Baijiatuan Village Gas Supply Station (Beijing, China). The TUNEL kit was purchased from Beyotime (Jiangsu, China). TNF-α, IL-6, and IL-1β enzyme-linked immunosorbent assay (ELISA) kits were purchased from Dakewe Biotech Company (Shenzhen, China). Analytically pure Na2SO3 and NaHSO3 were purchased from Beijing KangPuhui Technology Company (Beijing, China). Anti-ICAM1 antibody [YN1/1.7.4] (FITC) was purchased from Abcam (Shanghai, China).
4
Licorice-Derived Liquiritigenin Apoptosis
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Standard liquiritigenin of purity >98% was purchased from Aladdin Holdings Group Co., Ltd. (Shanghai, China). CP of over 98.5% purity (by HPLC) and the TUNEL kit were purchased from Beyotime Biotechnology (Shanghai, China). Annexin V-FITC/PI double staining apoptosis detection kit were obtained from BestBio (Shanghai, China). The antibodies used for Western blot analysis were as follows: anti-PGC-1α (Mouse, 1:1000, Catalog No.66369-1-Ig), anti-TFAM (Rabbit, 1:1000, Catalog No.22586-1-AP), anti-BCL-2 (Rabbit, 1:1000, Catalog No.26593-1-AP), anti-BAX (Mouse, 1:1000, Catalog No.60267-1-Ig), anti-β-actin (Mouse, 1:6000, Catalog No.66009-1-Ig), purchased from Protein Tech Group (Chicago, IL, USA), and anti-SIRT3 (Rabbit, 1:1000, Catalog No.ab189860), purchased from Abcam (Abcam, Cambridge, UK). Reagents related to cell culture such as culture medium, fetal bovine serum, and streptomycin and penicillin were purchased from Gibco (Shanghai, China).
5
Apoptosis Analysis in Retinal Organoids
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Retinal organoids were collected and prepared for cryosections. TUNEL kit (Beyotime Biotechnology) was suited for apoptosis analysis. Briefly, cryosections were fixed using 4% paraformaldehyde at room temperature for 30 min, then permeabilized in 1% Triton-X 100 for 15 min at 37°C. Then, sections were then incubated with TUNEL assay solution in the dark for 1 h at 37°C. The nuclei were stained with DAPI for 5 min. After washing three times with PBS, sections were mounted with fluorescence mounting medium and captured with a fluorescence microscope (Leica). ImageJ software was used to quantify DAPI-positive and TUNEL-positive areas. Data were shown as the percentage of TUNEL-positive areas.
6
Quantifying Apoptosis After Subarachnoid Hemorrhage
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Cell apoptosis was detected with a TUNEL kit (Beyotime, China) at 24 h after SAH in strict accordance with the manufacturer’s instructions. Briefly, the deparaffinized and rehydrated coronal brain slices were prepared as described above. After the brain slices were washed with PBS, they were incubated with TUNEL mixture for 1 h at room temperature, and then stained with DAPI. The results of TUNEL staining were observed and analyzed in the same way as FJC staining. The data are expressed as the ratio of TUNEL-positive cells to DAPI-positive cells.
7
Apoptosis Detection in Cerebral Cortex
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The brain tissue was dewaxed with xylene for 20 mins, rehydrated with a gradient of ethanol (from a high concentration to a low concentration) and permeabilized with proteinase K. The TUNEL kit was used to detect apoptosis in the cerebral cortex according to the manufacturer’s instructions (Beyotime, Shanghai, People's Republic of China). Apoptosis index (AI)=(TUNEL positive cells/total cells)×100%.
8
TUNEL Staining of Xenograft Sections
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Paraffined-embedded xenograft sections were subjected to TUNEL staining according to the manufacturer’s instructions. Briefly, paraffined-embedded xenograft sections were deparaffinized in xylene, rehydrated in graded ethanol, and washed with water before being treated with 20μg/mL Proteinase K (B600452, Sangon, Shanghai, China) for 15 min at 37 °C. Subsequently, they were washed with PBS 3 times and then stained with the TUNEL kit (C1086, Beyotime, Beijing, China) for 60 min at 37 °C in dark. After being stained with TUNEL solution, xenograft sections were counterstained with 4, 6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) for 5 min. The apoptotic cells were detected, and the results were observed using a fluorescence microscope (Leica DMi8, Wetzlar, Germany).
9
Histological Analysis of Kidney Tissue
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For H&E, Sirius red, Masson's trichrome and Periodic acid-Schiff (PAS) staining, tissues were isolated and fixed overnight at 4°C, and treated with 30% sucrose for cryoprotection. Sections of kidney were dewaxed with xylene and a series of descending ethanol gradients. Then sections were stained with Mayer's H&E (Jiancheng) or Sirius Red F3B and a saturated aqueous solution of picric acid (Solarbio, beijing, China), Masson (Jiancheng) or PAS kit (Solarbio), and observed with a Nikon Eclipse Ni-U microscope. For immunohistochemistry (IHC), paraffin sections were stained with anti-Nephrin (Abcam, Cambridge, UK), assessed by light microscopy and quantified using ImageJ (Wayne Rasband, US National Institutes of Health, Bethesda, MD, USA). For β-GAL, TUNEL and immunofluorescence staining, frozen tissue slices were stained with a β-GAL kit (Beyotime, Biotechnology, Shanghai, China), TUNEL kit (Beyotime), or anti-53BP1 (NOVUS, Abingdon, UK), respectively, photographed using an Olympus IX-71 fluorescence microscope and quantified using ImageJ. For DHE staining, 24 h before killing, mice received a 200 μL intravenous injection of dihydroethidium kit (Sigma-Aldrich, St Louis, MI, USA) at 25 mg/kg, and frozen tissue slices were photographed using an Olympus IX-71 fluorescence microscope and quantified using ImageJ.
10
Apoptosis Detection in Myocardial Tissues
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To detect the apoptotic cells in the myocardial tissues, TUNEL staining was conducted in accordance with the manufacturer’s protocol. The TUNEL kit was purchased from Beyotime (China). The tissues were sliced and the sections were dehydrated by gradient ethanol. Next, the slices were incubated with Triton X-100 (10%) for 1 hour at 37 °C, and TUNEL reaction reagent was added. 4’,6-diamidino-2-phenylindole was used to incubate the slices for 3–5 min at room temperature. After washing the slices, the TUNEL stain images were taken by an inverted fluorescence microscope (Leica, German).
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