TUNEL In Situ Apoptosis Kit (HRP-DAB Method)
中文名称:TUNEL原位细胞凋亡检测试剂盒(HRP-DAB显色法)
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- 应用: 细胞凋亡-DNA片段化
检测原理 | 细胞在发生凋亡时,会激活一些DNA内切酶,剪断核小体间的基因组DNA,暴露的3’-OH可在末端脱氧核酸转移酶(TdT)的催化下加上生物素标记的dUTP,辣根过氧化物酶(HRP)标记的Streptavidin (Streptavidin-HRP)可与之结合,在HRP的催化下通过DAB显色可观测到凋亡细胞,从而可通过普通光学显微镜检测到细胞的凋亡。由于正常的或正在增殖的细胞中几乎没有DNA的断裂,因而没有3'-OH形成,很少能够被标记。这就是TUNEL(TdT-mediated dUTP Nick-End Labeling)法检测细胞凋亡的原理。 |
检测方法 | DAB显色 |
样本类型 | 石蜡切片;冰冻切片;细胞爬片;细胞涂片 |
检测时间 | 3 hours |
检测仪器 | 普通光学显微镜 |
自备试剂 | 4%多聚甲醛(E-IR-R113),0.2% Triton X-100(E-IR-R122),3% H2O2(E-IR-R115),二甲苯,乙醇,DNase I,PBS(E-BC-R187),ddH2O,苏木素(E-IR-R120),中性树胶(E-IR-R118) |
保存温度 | 该产品可在-20°C避光条件下保存12个月。 |
有效期 | 12个月 |
运输条件 | 冰袋 |
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E-CK-A211 | Annexin V-FITC/PI Apoptosis Kit | 200Assays , 100Assays , 50Assays , 20Assays |
E-CK-A301 | Mitochondrial Membrane Potential Assay Kit (with JC-1) | 100Assays , 50Assays , 20Assays |
E-CK-A320 | One-step TUNEL In Situ Apoptosis Kit (Green, FITC) | 100Assays , 50Assays , 20Assays |
E-CK-A362 | Enhanced Cell Counting Kit 8 (WST-8/CCK8) | 500Tests , 100Tests , 500Tests×20 |
Q1:What is the difference between TUNEL-fluorescence and HRP-DAB TUNEL?
Color development of DAB (result of TUNEL-HRP) is observed with an ordinary microscope, the results of TUNEL nuclear staining were served with white light. As for TUNEL fluorescence, the results of TUNEL and nuclear staining were observed with different fluorescence channels seperately with better definition and resolution.
Q2:Is it avaliable to take a TUNEL assay with a microplate instead of a cell slides/smears?
(1) The slides can be sealed with anti-fluorescence quenching agent, but the microplate cannot. (2) The amount of reagents will be increased with 6-well plate. (3) The visual field will be decreased due to the smaller diameter of wells.
Q3:Can TUNEL assay kit be used for mouse samples?
There is no species limination in TUNEL assay kits.
Q4:If the frozen section have been fixed before freezing, do they still need to be fixed and incubated at room temperature for 30min?
If it has been fixed, it is not necessary to incubate and fix again, and subsequent experimental steps can be directly carried out.
Q5:Do samples need to be blocked when using the HRP-DAB TUNEL kit?
Yes they do. The blocking agent can be 3% H2O2. Peroxidase is present in many tissues, resulting in false positive results. Thus the endogenous peroxidase should be eliminated before staining. 3% hydrogen peroxide can react with peroxidase to eliminate endogenous peroxidase as a blocking agent.
Q6:Is the cell slides/smears for TUNEL assay able to be stored overnight after fixation?
Yes, soak in PBS and store at 4℃.
Q7:Why should I take the HRP-DAB TUNEL assay with shading light?
1. To avoid the effect of light on TdT enzyme activity. 2. Biotin molecules are easily decomposed by light, and the double bonds in biotin molecules are easily excited by light, and the decomposition produces substances such as ultraviolet light and active free radicals. These decomposition products will destroy the chemical binding between biotin and labeled molecules, thus affecting the accuracy and stability of experimental results.
Q8:Can the One-step TUNEL kit detect a sperm sample?
Yes, it can be used to test sperm samples.
Q9:What is the difference between TUNEL in-situ kit and TUNEL flow cytometry kit?
The main difference lies in the detection methods, for TUNEL in-situ kits, a fluorescence microscope is used for observing the results, as for TUNEL flow ytometry kit, the sample will be detected by a flow cytometer. On the other hand, TUNEL in-situ kit is more suitable for tissue sections and cell slides or smears, while TUNEL flow ytometry kit is more suitable for suspended cells and adherent cells.
Q10:Is there any TUNEL assay kit that can detect apoptosis in erythrocyte?
The detection of TUNEL is based on DNA fragmentation in late stage apoptosis, so it is not avaliable for erythrocyte as there is no nuclei or other organelles containing DNA in erythrocyte.