Formalin embedded tissues were sectioned and stained with Hematoxylin and Eosin (H&E). Frozen sections were fixed with 4% paraformaldehyde in PBS for 15 min. For staining of proliferating cells, or macrophage , the sections were incubated with anti-mouse Ki67 rabbit polyclonal antibody (Abcam, Cambridge, MA), anti-mouse F4/80 or CD68 rat polyclonal antibody (AbD serotec, Raleigh, NC) at 4°C overnight followed by incubation with corresponding secondary antibody, respectively. TUNEL assay was performed using TUNEL System (Promega Biosciences, LLC, San Luis Obispo, CA USA) according to the manufacturer’s protocol. The sections were imaged under laser scanning confocal microscope (Nikon Inc.), and the images were analyzed using NIS Elements software. DAPI nuclear stain was used to define the number of cells and the border of the tumor lesions (based on the density of nuclei). Fluorescent images (n=10-12) were captured and processed for regions of interest (ROI) using NIS elements software. To evaluate the macrophage distribution in various regions of the liver, the relative fluorescent intensities of F4/80 cells in ROI inside the tumor, tumor periphery (40-50 μm from the tumor border) and in the unaffected tissue were measured and normalized to DAPI signals (number of cells).
Tunel system
Manufactured by Promega
Sourced in United States
The TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) system is a laboratory technique used to detect and quantify apoptosis, or programmed cell death, in cells. The system employs enzymatic labeling of DNA strand breaks, allowing for the identification and visualization of cells undergoing apoptosis.
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Lab products found in correlation
Deadend colorimetric terminal, by Promega (2 mentions)
Protease k, by Merck Group (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Anti mouse f4 80, by Bio-Rad (1 mentions)
Laser scanning confocal microscope, by Nikon (1 mentions)
Deadend colorimetric tunel system, by Promega (1 mentions)
Lsm 510 confocal microscope, by Zeiss (1 mentions)
Annexin 5 fluorescein isothiocyanate fitc apoptosis detection kit, by Merck Group (1 mentions)
Eclipse ti s, by Nikon (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Vectasheild mounting medium with dapi, by Vector Laboratories (1 mentions)
Deadend fluorometric terminal, by Promega (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
One step tunel apoptosis assay kit, by Beyotime (1 mentions)
Glial fibrillary acidic protein (gfap), by Abcam (1 mentions)
Iba 1, by Abcam (1 mentions)
Tritc conjugated anti rabbit igg, by ZSGB-BIO (1 mentions)
Dnase 1, by Roche (1 mentions)
Confocal fluorescence microscope, by Leica (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
36 protocols using tunel system
1
Comprehensive Histological and Immunohistochemical Analysis
2
Apoptosis detection in tumor sections
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Terminal deoxynucleotidyl transferase (TdT) mediated-16-deoxyuridine triphosphate (dUTP) Nick-End Labelling (TUNEL) system (Promega, USA) was used to detect apoptosis in tissue sections. Tumor sections were de-paraffinized by xylene, washed in PBS, then rehydrated using serial concentrations of ethanol. Paraformaldehyde (4%) was used to fixed sections followed by washing in PBS and permeabilization using 20 μg/ml Proteinase K solution. Fragmented DNA was labeled by incubating sections with biotinylated dUTP in rTdT reaction mixture at 37°C for one hour and endogenous peroxidases were blocked using 0.3% hydrogen peroxide. Sections were incubated with streptavidin conjugated HRP at room temperature for 30 min. visualization of fragmented DNA was performed using hydrogen peroxide Followed by chromagen diaminobenzidine.
3
Quantifying Tumor Cell Apoptosis via TUNEL Assay
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Apoptosis in tumor cells was measured using the DeadEnd Colorimetric terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System
(Promega) according to the manufacturer's instructions. In brief, tumor sections
(4-μm thick) were incubated using TUNEL (DeadEnd™ Colorimetric TUNEL System,
Promega) for 2 h at 37°C. Finally, images were captured with a Zeiss LSM 510
confocal microscope (Germany) at 488 nm. The number and percentage of
TUNEL-positive cells were determined by counting 1×103 cells from six
random selected fields.
deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System
(Promega) according to the manufacturer's instructions. In brief, tumor sections
(4-μm thick) were incubated using TUNEL (DeadEnd™ Colorimetric TUNEL System,
Promega) for 2 h at 37°C. Finally, images were captured with a Zeiss LSM 510
confocal microscope (Germany) at 488 nm. The number and percentage of
TUNEL-positive cells were determined by counting 1×103 cells from six
random selected fields.
4
Cisplatin-Induced Apoptosis Evaluation
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Cells 1×103 cells/well were seeded in 6-well plates and incubated in a humid incubator at 37°C with 5% CO2 for 24 h, followed by 25 µg/ml cisplatin (cat. no. 479306; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) treatment at 37°C with 5% CO2 for 48 h. Cell apoptosis was assessed by the TUNEL system (Promega Corporation, Madison, WI, USA) and an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (EMD Millipore, Billerica, MA, USA), and then analyzed using flow cytometry (EPICS XL flow cytometer; Breckman Coulter, Inc., Brea, CA, USA) and a fluorescence microscope equipment with a digital camera. Three independent experiments were done, in triplicate.
5
Quantifying Apoptosis in Rat Cortex
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A DeadEnd™ Fluorometric Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP-biotin nick-end labeling (TUNEL) System (Promega, Madison, WI, USA) was used to confirm apoptosis in the rat cortex. TUNEL was performed according to the manufacturer’s instructions. Briefly, 100 μL of 20 μg/mL proteinase K was added to each slide to cover the tissue sections, and the sections were incubated for 10 minutes at room temperature. Then, the nuclei were stained with DAPI for 10 minutes. Finally, the sections were examined from the peri-lesion cortex with the fluorescence microscope (Nikon Eclipse Ti-S). Data were expressed as the ratio of TUNEL to total nuclei.
6
Cell Viability and DNA Fragmentation
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Cell viability was determined by using the CellTiter-Glo luminescent cell viability assay (Promega Corporation, Madison, WI, USA) according to the manufacturer’s directions. DNA fragmentation was determined by using the DeadEnd™ Fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System (Promega Corporation) according to the manufacturer’s directions. Nuclei were stained using Vectasheild mounting medium with DAPI (Vector Laboratories, Inc., Burlingame, CA, USA).
7
Tumor Apoptosis Assessment Protocol
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Tumor sections were stained with TUNEL system (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. Briefly, the tumor tissues from the four mouse groups were fixed in 10% formaldehyde at room temperature overnight, and then were embedded in paraffin. The sections were then deparaffinized with xylene, rehydrated and then treated with 3% hydrogen peroxide to quench the endogenous peroxidase activity. Subsequent antigen retrieval was performed by heating in citrate buffer solution (0.01 M) using a microwave oven. Sections were cut at 5 µm, dewaxed and rehydrated, washed with PBS, reacted with proteinase K (2 µl 50X proteinase K and 98 µl PBS) at room temperature for 20 min. Samples were labeled using TdT reaction mix and incubated for 1 h at 37°C in a humidified chamber (80–90%). Subsequently, 2X saline sodium citrate buffer (Ningxia Bauhinia Pharmaceutical Co., Ltd.) was added for 15 min to stop the reaction. Following hematoxylin staining, samples were mounted on to neutral balsam medium. Apoptotic cells were identified using dark brown nuclear staining (100 cells obtained from six random microscopic fields in each group) using a Nikon Eclipse 90i microscope (Nikon Corporation, Tokyo, Japan).
8
Measurement of Viral Vector-Induced Apoptosis
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Apoptosis of viral vector-transduced cells was measured using a DeadEnd Colorimetric terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) System (Promega) and One Step TUNEL Apoptosis Assay Kit (Beyotime) as described previously [17 (link)]. The number of stained cells that exhibit apoptotic-like morphology was assessed by counting cells from 10 randomly chosen fields per well.
9
Detecting Apoptosis in Breast Cancer Cells
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Apoptotic breast cancer cells were detected by TUNEL assay using the fluorometric TUNEL system (Promega, Madison, WI, USA) according to the protocol provided by the manufacturer. Briefly, 50 µL of rTdT incubation buffer was added to the permeabilized cells on slides, which were then incubated under humidified atmosphere for 60 min in darkness and rinsed three times with 2X saline-sodium citrate buffer. The slides were subsequently stained with DAPI and imaged under a fluorescence microscope (BX51, Olympus, Japan). TUNEL-positive cells were indicated by the emission of green fluorescence and the nuclei were indicated by their blue fluorescence.
10
Immunohistochemical Analysis of Brain Cell Markers
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After 3 days of DBI, the rat brains were perfused with cold PBS and quickly embedded in OCT medium (Sakura, USA), after which 8‐μm‐thick sections were prepared. The sections were subsequently fixed in cold acetone at 4°C for 5 min, blocked with 3% BSA at room temperature for 30 min, and then incubated with the following primary antibodies at 4°C overnight: Iba‐1 (1:500, Abcam, catalog number: ab5076), AQP4 (1:500, Cell Signaling Technology, catalog number: 59678T), CD31 (1:500, R&D Systems, catalog number: AF3628), and GFAP (1:500, Abcam, catalog number: ab48004). Then, the sections were incubated with Tritc‐conjugated anti‐rabbit IgG (1:100; Zsgb‐bio) at room temperature for 1 h in the dark. DAPI was used as a counterstain for 5 min. The negative controls were treated with PBS. Finally, the slices were visualized under a fluorescence microscope at ×200 magnification, and the positive cells in matched sections were counted using ImageJ software (version 1.48v, NIH).
Brain cell apoptosis was evaluated using a fluorometric terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) system (Promega, Madison, WI). Negative controls were prepared with the same procedures but without the primary antibody. The positive areas of polarized light microscopy with second harmonic generation (PSR) were quantified using ImageJ software.
Brain cell apoptosis was evaluated using a fluorometric terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) system (Promega, Madison, WI). Negative controls were prepared with the same procedures but without the primary antibody. The positive areas of polarized light microscopy with second harmonic generation (PSR) were quantified using ImageJ software.
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