According to our previous report (35 (link)), RGC apoptosis, as well as the protein expression of apoptosis-related cytokines, was detected mostly on day 1 post AOH. One day after AOH induction, rats were deeply anesthetized with 30 mg/kg pentobarbital sodium prior to cardiac perfusion with 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd.) for 24 h. The enucleated eyes were embedded in paraffin (65˚C) and sectioned at a thickness of 7 µm using a Leica DM2500 microtome (Leica Microsystems GmbH). TUNEL staining (37˚C; 1 h) of the retina was performed using a TUNEL assay kit (Promega Corporation), whereas the cell nuclei were stained with 50 mM DAPI (37˚C; 15 min, Vector Laboratories, Inc.). As a positive control, sections were incubated (room temperature; 10 min) with 0.5 µg/ml DNase I (Promega Corporation) before adding the equilibration buffer of the TUNEL assay kit. TUNEL-positive cells were observed and counted under fluorescence microscopy. The TUNEL-positive cells in each section were counted and quantified as per mm2 of the retina by using the image analysis program Image J. A total of six images on x20 magnification were used from two sections per animal.
Tunel assay kit
Manufactured by Promega
Sourced in United States
The TUNEL assay kit is a laboratory tool used to detect and quantify DNA fragmentation, a hallmark of apoptosis or programmed cell death. The kit provides reagents and protocols to label and visualize DNA strand breaks in fixed cells or tissue sections. It enables researchers to analyze and evaluate cellular death processes in various experimental systems.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Camxc 30, by Olympus (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Lsm 900 confocal microscope, by Zeiss (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Eclipse ti, by Nikon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Zen 2012 sp5, by Zeiss (2 mentions)
Rat anti mouse ki67 clone sola15 antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 anti rat secondary antibody, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (2 mentions)
Lsm 700, by Zeiss (2 mentions)
In situ cell death detection kit, by Promega (1 mentions)
Pe annexin 5 apoptosis detection kit, by BD (1 mentions)
87 protocols using tunel assay kit
1
Retinal Ganglion Cell Apoptosis Detection
2
Quantifying Cardiomyocyte Apoptosis via TUNEL
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The terminal deoxynucleotide transferase dUTP nick-end labelling (TUNEL) assay was used to detect the rate of cardiomyocyte apoptosis. NRVMs and peri-infarct cardiac tissue were prepared as described for immunofluorescence staining. After labelling with a secondary antibody, a TUNEL assay kit (Promega, USA) was used to stain apoptotic nuclei, according to the manufacturer's instructions.
3
Brain Tissue Infarct and Cell Death Assay
4
Quantifying Renal Apoptosis via TUNEL
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Apoptotic cells were detected using a TUNEL assay kit (Promega Corporation, Madison, WI, USA). Fresh kidney sections were fixed in 10% buffered formalin and embedded in paraffin, and 4-µm slices were stained using the in situ Cell Death Detection kit (Promega Corporation) according to the manufacturer's instructions. Three tissue sections from each sample were randomly selected and 10 microscopic fields per section were assessed by two independent observers. In each field, the nuclei were quantified and the percentage of TUNEL-positive nuclei was determined.
5
Apoptotic Activity of [10]-Gingerol
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The pro-apoptotic activity of [10]-gingerol was analysed by flow cytometry with the PE-Annexin-V Apoptosis Detection Kit (Becton, Dickinson, Franklin Lakes, NJ, USA) or using the TUNEL assay kit (Promega Corporation, USA) according to the manufacturer's instructions and as described in Supplementary methods.
6
Quantifying Cell Proliferation and Apoptosis
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For immunohistochemical analyses, explants and xenograft tumor tissues were processed, paraffin-embedded, and incubated with an antibody against Ki67 (#NB500, Novus, 1:100) or subjected to apoptosis assay using a TUNEL assay kit (#G3250, Promega Inc.) as described previously15 (link). Ki67 and TUNEL-positive cells were counted at ten arbitrarily selected fields at 40X magnification. The proliferation/apoptotic index (per 40X microscopic field) was determined as (number of Ki67/TUNEL-positive cells × 100)/total number of cells.
7
TUNEL Assay for Apoptotic DNA Fragmentation
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Fragmentation of DNA, a late event during apoptosis, was determined by enzymatic labelling of DNA strand breaks using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). TUNEL labeling was conducted using TUNEL assay kit (Promega, Madison, WI) on cells treated for 72 h with DMSO or 50 nM thioalbamide, according to the manufacturer’s instructions. Nuclear staining was performed by using 0.2 mg/mL 4′,6- diamidino-2-phenylindole (DAPI; Sigma Aldrich) and samples were analyzed with a fluorescent microscope (Olympus BX4 with CSV1.14 software using a CAMXC-30 for image acquisition).
8
TUNEL Assay for Apoptosis Evaluation
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The TUNEL staining was performed to detect cell apoptosis in tumor sections using a TUNEL assay kit according to the manufacturer’s instructions (Promega, Madison, WI, USA) and those of a previous study (24 (link)). Permount solution was used to dehydrate and mount the stained slides, and a BX43 light microscope (Olympus Corporation, Tokyo, Japan) was used to visualize effect of the permount solution on the slides.
9
Synthesis and Characterization of Compounds 5, 8, 9
10
Apoptosis Detection in Untreated Mice
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Fixed sections obtained from untreated mice were fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100. TUNEL assay kit (Promega) was used to detect apoptotic cells and DAPI was used to stain the nuclei.
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