TUNEL staining was performed to detect cellular apoptosis in the gastrocnemius sections after femoral artery ligation according to the TUNEL staining kit manufacturer’s instructions (Abcam, Cambridge; United Kingdom; cat no. ab66110). In brief, tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 mm transverse sections. Paraffin sections of the aorta were stained with an in situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit (ab66110, Abcam). 4′,6′-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. HUVECs were stimulated with high glucose (25 mM; Sigma–Aldrich; 20 (link)) or 3-IAId (0.5 mM; Sigma–Aldrich; 5 (link)) for 24 h and then subjected to TUNEL staining at 37°C for 30 min. The number of TUNEL-positive nuclei was analyzed using ImageJ software (NIH, Bethesda, MD, United States). The TUNEL-positive cell number per field for each sample was the average of 6 random fields.
Tunel staining kit
Manufactured by Abcam
Sourced in United States, United Kingdom
The TUNEL staining kit is a laboratory tool used to detect and visualize DNA fragmentation, a key feature of apoptosis or programmed cell death. The kit provides the necessary reagents and protocols to perform the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay, which labels the free 3'-hydroxyl termini of DNA strand breaks.
Automatically generated - may contain errors
Lab products found in correlation
Accuri c6, by BD (2 mentions)
Accuri c6 flow cytometer, by BD (2 mentions)
Dfc420 camera, by Leica (2 mentions)
High glucose, by Merck Group (1 mentions)
3 iaid, by Merck Group (1 mentions)
Vimentin, by Abcam (1 mentions)
Axio imager 2 microscope, by Zeiss (1 mentions)
Tunel staining kit, by Roche (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Biorad gel system, by Bio-Rad (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
7 protocols using tunel staining kit
1
TUNEL Assay for Apoptosis Detection
2
Apoptosis Detection by TUNEL Assay
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Cells were treated with DMSO or isoplumbagin for 12 h. After fixing with 1% (v/v) paraformaldehyde for 15 min, cells were washed with PBS two times and added to 70% (v/v) ethanol for 30 min on ice. After that, cells were stained by the usage of TUNEL staining kit (Abcam, #ab66108, Cambridge, MA, USA) according to the manufacturer’s instructions. Briefly, cells were incubated with DNA labeling solution for 60 min at 37 °C, followed by the addition of PI/RNase A solution for 30 min. TUNEL-labeled cells were evaluated by BD Accuri™ C6 flow cytometer, and the data were analyzed using BD Accuri C6 Software.
3
Quantifying Hippocampal Apoptosis in Rats
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The other half of the forebrains (n=6 rats per group) at 24 h after reperfusion was embedded in optimal cutting temperature compound (OCT) (Tissue-Tek), sectioned into 5-μm slides, and stained with a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) staining kit (Abcam, U.S.A.). The hippocampal apoptotic cells were counted. Cell nuclei were stained with DAPI. All fluorescent images were examined using a Leica DM3000 microscope and recorded using a DFC 420 camera (Leica, Germany) [21 (link)].
4
Reconstructed Skin Histology and Apoptosis
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Reconstructed skin equivalents from the above groups were in turn fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 6 μm serial slices. Histological features were determined using hematoxylin and eosin (H&E) staining. Apoptotic cells were detected using TUNEL staining kit (Abcam, UK) in accordance with the manufacturer's instructions. Cells with the positive expressions of vimentin and CK10 (Abcam, UK) were measured using IHC analysis. Positively stained cells were counted in five randomly selected fields through a double-blind microscopic evaluation by two independent observers. Simultaneously, the survival of different SKPs was tracked using fluorescent labeling.
5
Apoptosis Analysis in Myocardial Tissues
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Hearts were collected from five mice in the control and DOX groups, respectively. Frozen heart tissues were cut into 5 μm thick sections, and H9c2 cells were cultured on cover glass. H9c2 cells were transfected with ORM1 virus, Nrf2 siRNA, or the corresponding control. After 48 h of transfection, cells were stimulated with DOX for 24 h and fixed with 4% paraformaldehyde. Apoptosis in myocardial tissues was analyzed using a TUNEL staining kit (Roche, Switzerland), and H9c2 cells were analyzed using a different TUNEL staining kit (Abcam, UK) according to the manufacturer's instructions. Fluorescence images were examined using a Zeiss Axio Imager 2 microscope (Zeiss, Germany).
6
Apoptosis Evaluation by TUNEL Assay
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Cells were treated with DMSO or isoplumbagin for 12 h. After fixed with 1% (v/v) paraformaldehyde for 15 min, cells were washed with PBS for two times and added with 70% (v/v) ethanol for 30 min on ice.
After that, cells were stained by the usage of TUNEL staining kit (Abcam, #ab66108, Cambridge, MA, USA) according to the manufacturer's instructions. Briefly, cells were incubated with DNA labeling solution for 60 min at 37°C, followed by addition of PI/RNase A solution for 30 min. TUNEL-labeled cells were evaluated by BD Accuri™ C6 flow cytometer and the data were analyzed using BD Accuri C6 Software.
After that, cells were stained by the usage of TUNEL staining kit (Abcam, #ab66108, Cambridge, MA, USA) according to the manufacturer's instructions. Briefly, cells were incubated with DNA labeling solution for 60 min at 37°C, followed by addition of PI/RNase A solution for 30 min. TUNEL-labeled cells were evaluated by BD Accuri™ C6 flow cytometer and the data were analyzed using BD Accuri C6 Software.
7
Western Blot and Cell Viability Analysis
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Total cell lysate was prepared in RIPA buffer supplemented with 1 mM PMSF. Protein concentrations were determined by the Bradford assay. Equal amount of proteins were loaded and separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in TBS-0.1% Tween 20 (TBST)/5% (w/v) milk for 2 h at room temperature. The membranes were then incubated with anti-PIK3C2A or antiβ-actin (Cell Signaling Technology, USA). Proteins on the membranes were detected using the ECL Kit (Thermo Scientific, USA) after washing in TBST, and the blots were quantified with Bio-Rad Gel system (Bio-Rad, USA).
Cell Counting Kit Assay 882R-NC, 882R-OE, and 882R-KD cells were cultured in a 96-well plate under regular conditions. At 24, 48, 72, and 96 h, 10 μl cell counting kit-8 (CCK8, Dojindo Laboratories, Japan) was added and incubated for 2 h at 37 °C in a humidified incubator. Optical density value was measured at 450 nm. IC50 were calculated by Logit method.
TUNEL Staining 882R-NC, 882R-OE, and 882R-KD cells were cultured on coverslips. Cells were harvested and fixed with 4% paraformaldehyde. Cell apoptosis analysis was performed using a TUNEL Staining Kit (Abcam, USA). Cell nuclei were stained with DAPI. All fluorescent images were examined using a Leica DM3000 microscope and photographed using a DFC 420 camera (Leica, Germany).
Cell Counting Kit Assay 882R-NC, 882R-OE, and 882R-KD cells were cultured in a 96-well plate under regular conditions. At 24, 48, 72, and 96 h, 10 μl cell counting kit-8 (CCK8, Dojindo Laboratories, Japan) was added and incubated for 2 h at 37 °C in a humidified incubator. Optical density value was measured at 450 nm. IC50 were calculated by Logit method.
TUNEL Staining 882R-NC, 882R-OE, and 882R-KD cells were cultured on coverslips. Cells were harvested and fixed with 4% paraformaldehyde. Cell apoptosis analysis was performed using a TUNEL Staining Kit (Abcam, USA). Cell nuclei were stained with DAPI. All fluorescent images were examined using a Leica DM3000 microscope and photographed using a DFC 420 camera (Leica, Germany).
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