Formalin-fixed tissue samples were embedded in paraffin and then the paraffin-embedded specimens were cut into serial sections (4-mm thick). Primary tumors, heart, liver, spleen, lung and kidney sections were stained with hematoxylin and eosin, and tumor specimens were immunostained with a Ki-67 (8D5) mouse mAb (1:400, Cell Signaling Technology), an eEF2K antibody (1:500, Abcam), an LC3B (D11) rabbit mAb (1:3200, Cell Signaling Technology), a Bcl-xL (54H6) rabbit mAb (1:300, Cell Signaling Technology) and a cleaved caspase-3 (Asp175) (D3E9) rabbit mAb (1:250, Cell Signaling Technology) at room temperature for 1 h in a humidified chamber. Sections were rinsed with PBS and exposed to appropriate species-specific secondary antibodies for 30 min. Finally, each section was exposed to 3,3’-diaminobenzidine (DAB) solution (KeyGen Biotechnology, China) for 3-5 min after they were rinsed with PBS. To evaluate the apoptotic response in tumor tissues, Colorimetric TUNEL Apoptosis Assay Kits (Beyotime) were used. The sections were deparaffinized, rehydrated through graded alcohols to water, treated with 20 μg/ml proteinase K (37 °C, 20 min) and then washed in 1x Equilibration Buffer. The TUNEL assay was then performed according to the instructions from the manufacturer. Images were captured via microscopy (Carl Zeiss, Germany).
Colorimetric tunel apoptosis assay kit
Manufactured by Beyotime
Sourced in China
The Colorimetric TUNEL Apoptosis Assay Kit is a laboratory tool designed to detect and quantify apoptosis, a type of programmed cell death, in cell samples. The kit uses the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) method to label and identify DNA fragments, a hallmark of apoptosis. The colorimetric detection system allows for the quantification of apoptotic cells in the sample.
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Lab products found in correlation
One step tunel apoptosis assay kit, by Beyotime (7 mentions)
Goat serum, by Boster Bio (3 mentions)
Ki67 antibody, by Bioss Antibodies (3 mentions)
Polink 1 hrp dab detection system one step polymer detection system, by ZSGB-BIO (3 mentions)
Optical microscope, by Olympus (3 mentions)
Eclipse 80i, by Nikon (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Masson s trichrome stain kit, by Solarbio (2 mentions)
Dako real envision kit, by Agilent Technologies (2 mentions)
Immunol staining fix solution, by Beyotime (2 mentions)
Proteinase k, by Beyotime (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Microscopy, by Zeiss (1 mentions)
Ki 67 8d5 mouse mab, by Cell Signaling Technology (1 mentions)
Lc3b d11 rabbit mab, by Cell Signaling Technology (1 mentions)
Bcl xl 54h6 rabbit mab, by Cell Signaling Technology (1 mentions)
3 3 diaminobenzidine dab solution, by Keygen Biotech (1 mentions)
Vascular endothelial growth factor vegf, by Abcam (1 mentions)
Penicillin streptomycin pen strep, by Thermo Fisher Scientific (1 mentions)
102 protocols using colorimetric tunel apoptosis assay kit
1
Immunohistochemical Analysis of Tumor Markers
2
Tumor Cell Line Characterization Protocol
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The VX2 tumor cell line was purchased from Jennio Biotech (China). HepG2 and Huh7 cell lines, LO2 cell lines, and special culture media were obtained from iCell (China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin (Pen-Strep), and trypsin-EDTA solution were supplied by Gibco (USA). Hexa-histidine (His6) was synthesized by Zhuantai Biocom (China). All chemicals, including 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), polyvinylpyrrolidone (PVPON, Mw∼58k), zinc nitrate hexahydrate, sodium hydroxide, and hydrochloric acid, were provided by Aladdin (China). Cell counting kit-8 (CCK-8) was purchased from GLPBIO (USA). Live/dead cell staining kits were obtained from APExBIO (USA). The Annexin V-Fluorescein Apoptosis Assay Kit was supplied by Multisciences (Beijing, China). DOX, crystal violet ammonium oxalate, methyl thiazolyl tetrazolium (MTT), and phosphate-buffered saline (PBS) were purchased from Solarbio (China). Actin-Tracker Green-488, Hoechst 33342, and colorimetric TUNEL Apoptosis Assay Kits were provided by Beyotime (China). Lipiodol was obtained from Guerbet (France). Ki-67 antibodies and vascular endothelial growth factor (VEGF) were obtained from Abcam (Cambridge, UK).
3
TUNEL Assay for Apoptosis Detection
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Cell apoptosis was measured using Colorimetric TUNEL Apoptosis Assay Kits (Beyotime, China). Cells were washed with PBS twice, fixed by 4% paraformaldehyde, immersed by 0.1% Triton X-100, and blocked by endogenous peroxidase blocking solution. The slides were then washed, incubated with a mix solution composed of the enzyme terminal deoxynucleotide transferase (TdT) and biotinylated (Bio-16) dUTP in TdT, and stopped with a stop buffer. Then slides were incubated with a streptavidin–HRP conjugate, stained with DAB buffer, washed, and observed via microscope.
4
Evaluating Apoptosis in Gonads
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TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique was applied to evaluate the apoptotic response of TTX administration in the ovary and testis. Reactions were performed on Sects. (5 µm) of 4% Paraformaldehyde (PFA)-fixed (overnight at 4 °C) and paraffin-embedded ovaries and testes. Apoptotic cells with DNA breaks were detected using Colorimetric TUNEL Apoptosis Assay Kits (#C1091, Beyotime Institute of Biotechnology, Shanghai, China). TUNEL assay was then performed according to the instructions by the manufacturer. Images were used a Nikon ECLIPES 80i light microscope (Nikon, Japan). The apoptosis signal ratio of gonads was a statistical analysis of the three different areas of each sample under TUNEL staining by using ImageJ (NIH, USA) and Graph Prism8.0 (San Diego, USA) as described before.
5
Apoptosis Evaluation in Hermaphroditic Gonad Development
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Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling technique was applied to evaluate the apoptotic response of EPOs in the testicular hermaphroditic stage. Reactions were performed on sections (5 µm) of 4% Paraformaldehyde (PFA)-fixed (overnight at 4°C) and paraffin-embedded L. polyactis’ ovaries and testes at 80 dph. Apoptotic cells with DNA breaks were detected using Colorimetric TUNEL Apoptosis Assay Kits (#C1091, Beyotime Institute of Biotechnology, Shanghai, China) and In Situ Cell Death Detection Kit, TMR red (#12156792910, Roche, Indianapolis, IN, USA). TUNEL assay was then performed according to the instructions by the manufacturer. For TMR red kit, DNA was stained with DAPI. Images were captured using a Zeiss light microscope Axio Imager 2 and Zeiss confocal microscope LSM710. Image processing and analysis were performed using AxioObserver (Carl Zeiss Microscopy GmbH, Jena, Germany) and ZEN software.
6
Colorimetric TUNEL Apoptosis Assay
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The TUNEL assay was conducted using Colorimetric TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Briefly, after incubation of apatinib for 48 h, A549 and H1299 cells were fixed, permeabilized, and then inactivated endogenous peroxidase by 0.3% H2O2 for 20 min. Subsequently, the cells were incubated with a mix solution containing enzyme terminal deoxynucleotide transferase (TdT) and biotinylated (Bio-16) dUTP in TdT buffer at 37 °C for 60 min and the nuclei were stained with diaminobenzidine for 10 min and counterstained with hematoxylin. Finally, the apoptosis cells were visualized by an invert microscope.
7
TUNEL Assay for Brain Apoptosis
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Cellular apoptosis in the brain tissue from different treatment groups was analyzed by TUNEL assay according to the manufacturer's instructions of a commercial kit (Colorimetric TUNEL Apoptosis Assay kit; C1091; Beyotime Institute of Biotechnology, Shanghai, China). Briefly, brains were embedded into paraffin and fixed with 10% formaldehyde at 27°C for 20 min. Following, brains were cut into 5-µm sections and incubated with 1× biotin-labeled deoxyuridine triphosphate (Beyotime Institute of Biotechnology, Haimen, China) at 37°C for 1 h. Samples were incubated with streptavidin-horseradish peroxidase (HRP) at 37°C for 30 min and 0.05% 3′-diaminobenzidine development solutions at 25°C for 10 min in sequential order. Brain slides were examined microscopically (light microscope; magnification, ×200) and imaged with a high-resolution digital camera (Nikon Eclipse 80i; Nikon Corporation, Tokyo, Japan).
8
Proliferation and Apoptosis Assays
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EdU and TUNEL assays were performed using the BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 488 and Colorimetric TUNEL Apoptosis Assay Kit (all from Beyotime), respectively, in accordance with the manufacturer’s protocol. The number of EdU or TUNEL-positive cells was counted in six fields randomly, and the proliferation/apoptosis index for each field was calculated as the percent of positive cells relative to the total cells.
9
Wogonin Modulates LPS-Induced Apoptosis
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TUNEL assay was carried out to assess the apoptosis of LPS-induced A549 cells with or without wogonin treatment by Colorimetric TUNEL Apoptosis Assay Kit (Beyotime, Shanghai, China) based on the operation guidelines. Following phosphate buffer saline (PBS) washing for twice and fixation by 4% paraformaldehyde for 0.5 h, 0.3% hydrogen peroxide in PBS was used to incubate cells for another 20 min at room temperature. Cells were then treated by diaminobenzene (DAB) for 10 min and counterstained by hematoxylin (Solarbio, Beijing, China) for 30 s. The apoptotic-positive cells were observed by a fluorescence microscope (Olympus Corporation) and the apoptotic rate was qualified by Image-J software (NIH, Bethesda, MD, USA).
10
Histological and Biochemical Liver Assessment
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The left lobe of mouse liver was placed in 10% formaldehyde, dehydrated and then embedded in paraffin blocks. It was sectioned to 4 µm thickness for staining using Masson's Trichrome Stain Kit and Picrosirius Red from Solarbio Technology Co., Ltd. Moreover, we also used the paraffin section for dewaxing to water and antigen repair. After the liver slices were incubated with primary antibody, we used UltraSensitive™ SP (Mouse/Rabbit) IHC Kit from Maixin Biotech. Co., Ltd for staining. We used the TUNEL staining method by using the Colorimetric TUNEL Apoptosis Assay Kit (Beyotime Biotechnology) to observe the apoptosis of liver tissue. We randomly selected the field of view to take pictures using upright transmission fluorescence 162 microscope (Olympus) and analysed the photos using Image‐Pro Plus Version 6.0 (Media 163 Cybernetics, Inc American). Apart from these, we used the alanine aminotransferase assay kit and aspartate aminotransferase assay kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) for ALT and AST content determination, respectively.
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