TUNEL-roche使用流程
TUNEL细胞凋亡试剂盒内容及操作步骤
TUNEL细胞凋亡试剂盒内容及操作步骤--------------------------------------------------------------------------------凋亡细胞的原位末断转移酶标记法（TUNEL法）细胞凋亡的多步骤机制作用的最终环节是；细胞内源性核酸内切酶的激活而导致核染色质DNA双链的断裂。
大量DNA片段暴露出的3 羟基在末断转移酶(terminal deoxynucleotidyl transferase, TdT)或DNA多聚酶的作用下，与生物素或地高辛标记的核苷酸结合，最终借助与卵白素或抗地高辛抗体结合的荧光素或HRP，使凋亡细胞被特异性地标记和显示出来。
TUNEL细胞凋亡试剂盒由美国罗氏公司提供试剂1：酶浓缩溶液（Enzyme Solution）试剂2：标记溶液（Lable Solution）试剂3：转化剂-POD（Converter-POD）酶标记抗荧光素抗体（即用型）试验所需其它试剂：非石蜡切片:·冲洗缓冲液:磷酸盐缓冲液（PBS）·阻断溶液:0.3%H2O2甲醇溶液·固定溶液:4%多聚甲醛（溶剂pH7.4新鲜配制的PBS溶液）·渗透液:0.1%Triton甔-100（溶于新鲜配制的0.1%枸橼酸钠溶液）Triton X-100(聚乙二醇辛基苯基醚)名：曲拉通X-100，乳化剂OP分子式：C34H62O11石蜡切片:·二甲苯和乙醇（100%、95%、90%、80%、70%用蒸馏水稀释）·冲洗缓冲液:磷酸盐缓冲液（PBS）·蛋白酶K 工作液10~20mg/ml溶于10mM Tris/HCl（pH7.4~7.8）根据需要选择:·渗透液:0.1%TritonX-100（溶于新鲜配制的0.1%枸橼酸钠溶液）·胃酶溶液（0.25%-0.5%溶于HCl ，pH2）或胰酶·0.1M枸橼酸缓冲液，pH6，微波修复材料：微波炉，微波输出功率850W-2000W2 .选用中性甲醇固定的活检及实验动物标本，常规脱水，二甲苯透明，石蜡包埋。
TUNEL细胞凋亡试剂盒内容及操作步骤精
TUNEL细胞凋亡试剂盒内容及操作步骤精TUNEL（Terminal deoxynucleotidyl transferase dUTP nick end labeling）细胞凋亡试剂盒是一种常用的检测细胞凋亡的方法。
本文将介绍TUNEL细胞凋亡试剂盒的内容及操作步骤。
1.引物溶液：含有dUTP（脱氧尿苷三磷酸）的荧光标记的核苷酸。
2.终止缓冲液：用于停止反应和洗涤。
3. TUNEL酶溶液：含有DNA连接酶（terminal deoxynucleotidyl transferase），用于在DNA断裂的末端添加dUTP标记。
4.正对照组：包括经过DNA内切酶处理的组织切片或细胞，用于确定试剂盒的反应情况。
步骤一：取材将需要检测的组织切片或细胞标本取出，放入离心管或培养皿中。
步骤二：固定用4%的冷乙醇或4%的帕拉醛对组织切片或细胞标本进行固定，固定时间为10-30分钟。
步骤三：洗涤用PBS（磷酸盐缓冲液）洗涤固定的样本3次，每次洗涤5分钟。
步骤四：蛋白酶消化对组织切片或细胞标本进行蛋白酶消化，以去除背景蛋白质干扰。
消化时间和消化酶的浓度需要根据实验条件进行优化。
步骤五：洗涤用PBS洗涤样本3次，每次洗涤5分钟。
步骤六：加入TUNEL酶溶液将TUNEL酶溶液滴加到样本上，保持湿润。
反应时间为1-2小时，可以根据实验需要进行优化。
步骤七：终止反应加入终止液停止反应，并用PBS洗涤样本3次，每次洗涤5分钟。
步骤八：显微镜观察将样本用荧光或显色试剂染色，然后用荧光显微镜或普通显微镜观察，并拍摄照片。
步骤九：分析数据使用图像分析软件对显微镜拍摄的照片进行图像分析，在感兴趣区域计算标记的细胞数量，并计算相对细胞凋亡率。
值得注意的是，在进行TUNEL细胞凋亡试剂盒检测时，需要根据实验需要进行优化，如反应时间、荧光染色时间、荧光强度测量等。
此外，为了准确地判断细胞中的凋亡现象，可以与其他细胞凋亡指标一起使用，如Annexin V染色、Caspase活性检测等。
翻译好的 罗氏公司Tunel试剂盒操作说明书
罗氏(Roche)公司Tunel试剂盒操作说明书(In situ cell death detection kit-POD法)一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）Enzyme Solution 酶溶液：TdT 10×、2号（紫盖）Label Solution标记液：荧光素标记的dUTP 1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5 μl 20×DAB+1μL 30%H2O2+94 μl PBS）、Proteinase K工作液（10-20 μg/ml in 10 mM Tris/HCl，pH 7.4-8）或细胞通透液（0.1% Triton X-100 溶于0.1% 柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml–3 U/ml in 50 mM Tris-HCl，pH 7.5，10 mM MgCl2，1 mg/ml BSA）等。
TUNEL染色
相关疾病：•脱水产品名称：罗氏(Roche)公司Tunel试剂盒英文名称：Tunel产品货号：11684817910产品规格：50T试剂级别：试剂级产品产地：USA产品商标：RocheIn situ cell death detection kit-POD法一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP 在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含TdT 10×、荧光素标记的dUTP 1×、标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5 μl 20×DAB+1μL 30%H2O2+94 μl PBS）、Proteinase K工作液（10-20 μg/ml in 10 mM Tris/HCl，pH 7.4-8）或细胞通透液（0.1% Triton X-100 in 0.1% sodium citrate，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl，pH 7.5，10 mM MgCl2，1 mg/ml BSA）等。
TUNEL细胞凋亡检测试剂盒罗氏
TUNEL细胞凋亡检测试剂盒罗⽒For research purposes only. Not for use for in vitro diagnosticprocedures for clinical diagnosis.In Situ Cell Death Detection Kit, PODKit for immunohistochemical detection and quantification of apop-tosis (programmed cell death) at single cell level, based on labeling of DNA strand breaks (TUNEL technology): Analysis by light microscopy.Cat. No. 1 684 817Store at ?15 to ?25°C 1 Kit (50 tests)Instruction ManualVersion 3, January 20031. Preface1.1Table of contentsP reface(2)1.1.1Table of contents (2)(3)1.2 Kitcontents(5)2. Introduction2.1Product overview (5)(8)2.2 Backgroundinformation3. Procedures and required materials (10)3.1Flow chart (10)3.2Preparation of sample material (11)3.2.1Adherent cells, cell smears and cytospin preparations (11)(12)sections3.2.2 Tissue3.2.2.1 Treatment of paraffin-embedded tissue (12)3.2.2.2Treatment of cryopreserved tissue (14)3.3Labeling protocol (15)3.3.1 Before you begin (15)3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations, and tissues (16)3.3.3 Labeling protocol for difficult tissue (17)(18)conversion3.4 Signal(19)4. Appendix(19)4.1 Trouble-shooting(22)4.2 References(23)4.3 Relatedproducts1.2 KitcontentsCaution The Label solution contains cacodylate, toxic by inhalation and swal-lowed, and cobalt dichloride, which may cause cancer by inhalation.Avoid exposure and obtain special instructions before use.When using do not eat, drink or smoke. After contact with skin, wash immediately with plenty of water. In case of accident or if you feelunwell seek medical advice immediately (show label where possible). Collect the supernatants from the labeling reactions in a tightly closed,non-breakable container and indicate contents. Discard as regulatedfor toxic waste.Kit contents Please refer to the following table for the contents of the kit.Vial/CapLabel Contents1 blue Enzyme Solution?Terminal deoxynucleotidyl transferasefrom calf thymus (EC 2.7.7.31), recom-binant in E. coli, in storage buffer10× conc.5×50l2 violet Label Solution?Nucleotide mixture in reaction buffer1×conc.5 × 550 l3 yellow Converter-POD?Anti-fluorescein antibody, Fab frag-ment from sheep, conjugated withhorse-radish peroxidase (POD)Ready-to-use3.5mlAdditional equipment required In addition to the reagents listed above, you have to prepare several solutions. In the table you will find an overview about the equipment which is needed for the different procedures.Detailed information is given in front of each procedure.Procedure Equipment Reagents Preparation of sample material (section 3.2)Adherent cells, cell smears and cytospinpreparations (section3.2.1)?Cryopreserved tissue (section 3.2.2.2)?Washing buffer: Phosphate buffered saline(PBS*)Blocking solution: 3% H2O2 in methanolFixation solution: 4% Paraformaldehyde inPBS, pH 7.4, freshly prepared ?Permeabilisation solution: 0.1% Triton X-100 in 0.1% sodium citrate, freshly pre-pared (6)Paraffin-embedded tissue (section 3.2.2.1)?Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in double distilled water)?Washing buffer: PBS*Proteinase K*, nuclease, working solution: [10-20 µg/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatments Permeabilisation solution: (0.1% Triton1) X–100, 0.1% sodium citrate) , freshly prepared Pepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free0.1 M Citrate buffer, pH 6 for microwave irradiationLabeling protocol (section 3.3)Positive control (section 3.3.1)?Micrococcal nuclease or ?DNase I, grade I *Adherent cells, cell smears, cytospin preparations, and tissues (section 3.3.2) ?Parafilm orcoverslipsHumidifiedchamberWashing buffer: PBS*Difficult tissue (section 3.3.3)?Plastic jarMicrowaveHumidifiedchamberCitrate buffer, 0.1 M, pH 6.0.Washing buffer: PBS*Tris-HCl, 0.1 M pH 7.5, containing 3% BSA*and 20% normal bovine serumSignal conversion (section 3.4)Humidified chamber Parafilm or coverslip Washing buffer: PBS*DAB Metal Enhanced Substrate Set* or alternative POD substrates Mounting medium for light microscopy1.2 Kitcontents,continued2. Introduction2.1Product overviewTest principle Cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments (mono- and oligonu-cleosomes) as well as single strand breaks (“nicks”) in high molecularweight DNA.Those DNA strand breaks can be identified by labeling free 3’-OH ter-mini with modified nucleotides in an enzymatic reaction.Fig. 1: Test principleApplication The In Situ Cell Death Detection Kit is designed as a precise, fast and simple, non-radioactive technique to detect and quantify apoptotic celldeath at single cell level in cells and tissues. Thus, the In Situ CellDeath Detection Kit can be used in many different assay systems.Examples are:Detection of individual apoptotic cells in frozen and formalin fixedtissue sections in basic research and routine pathology.Determination of sensitivity of malignant cells to drug induced apo-ptosis in cancer research and clinical oncology.Typing of cells undergoing cell death in heterogeneous populationsby double staining procedures (6).Specificity The TUNEL reaction preferentially labels DNA strand breaks gener-ated during apoptosis. This allows discrimination of apoptosis fromnecrosis and from primary DNA strand breaks induced by cytostaticdrugs or irradiation (3, 4).Test interference False negative results: DNA cleavage can be absent or incomplete in some forms of apoptotic cell death (37). Sterical hindrance such asextracellular matrix components can prevent access of TdT to DNAstrand breaks. In either case false negative results can be obtained.False positive results: Extensive DNA fragmentation may occur in latestages of necrosis (4, 38).DNA strand breaks may also be prominent in cell populations withhigh proliferative or metabolic activity. In either case false positiveresults may be obtained. To confirm apoptotic mode of cell death, themorphology of respective cells should be examined very carefully.Morphological changes during apoptosis have a characteristic pattern.Therefore evaluation of cell morphology is an important parameter insituations where there is any ambiguity regarding interpretation ofresults.Sample material?Cytospin and cell smear preparationsAdherent cells cultured on chamber slides (31)Frozen or formalin-fixed, paraffin-embedded tissue sections (1, 25,26, 29, 30, 32–34, 36, 39)Assay time2–3 hours, excluding culture, fixation and permeabilisation of cells and preparation of tissue sections. Number of tests The kit is designed for 50 tests.Kit storage/ stability The unopened kit is stable at ?15 to ?25°C through the expiration date printed on the label. Reagent Storage and stabilityTUNEL reaction mixture The TUNEL reaction mixture should be pre-pared immediately before use and shouldnot be stored.Keep TUNEL reaction mixture on ice untiluse.Converter-POD Once thawed the Converter-POD solutionshould be stored at 2–8°C (maximum stabil-ity 6 months).Note: Do not freeze!Advantage Please refer to the following table.Benefit FeatureSensitive Detection of apoptotic cell death at singlecell level at very early stages (1, 2, 6).Specific Preferential labeling of apoptosis versusnecrosis (3, 4).Fast Short assay time (2-3 h).Convenient?Reagents are provided in stable, opti-mized form.No dilution steps required.Flexible?Suitable for fixed cells and tissue. Thisallows accumulation, storage and trans-port of samples (2, 5).Double staining enables identification oftype and differentiation state of cellsundergoing apoptosis (6).Function-tested Every lot is function-tested on apoptoticcells in comparison to a master lot.2.2 BackgroundinformationCell death Two distinct modes of cell death, apoptosis and necrosis, can be distin-guished based on differences in morphological, biochemical andmolecular changes of dying cells.Programmed cell death or apoptosis is the most common form ofeukaryotic cell death. It is a physiological suicide mechanism that pre-serves homeostasis, in which cell death naturally occurs during normaltissue turnover (8, 9). In general, cells undergoing apoptosis display acharacteristic pattern of structural changes in nucleus and cytoplasm,including rapid blebbing of plasma membrane and nuclear disintegra-tion. The nuclear collapse is associated with extensive damage tochromatin and DNA-cleavage into oligonucleosomal length DNA frag-ments after activation of a calcium-dependent endogenous endonu-clease (10, 11). However, very rare exceptions have been describedwhere morphological features of apoptosis are not accompanied witholigonucleosomal DNA cleavage (37).Apoptosis Apoptosis is essential in many physiological processes, includingmaturation and effector mechanisms of the immune system (12, 13),embryonic development of tissue, organs and limbs (14), developmentof the nervous system (15, 16) and hormone-dependent tissueremodeling (17). Inappropriate regulation of apoptosis may play animportant role in many pathological conditions like ischemia, stroke,heart disease, cancer, AIDS, autoimmunity, hepatotoxicity and degen-erative diseases of the central nervous system (18–20).In oncology, extensive interest in apoptosis comes from the observa-tion, that this mode of cell death is triggered by a variety of antitumordrugs, radiation and hyperthermia, and that the intrinsic propensity oftumor cells to respond by apoptosis is modulated by expression ofseveral oncogenes and may be a prognostic marker for cancer treat-ment (21).Identification of apoptosis Several methods have been described to identify apoptotic cells (22– 24). Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detec-tion of apoptosis by the typical “DNA ladder“ on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation.This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucle-otidyl transferase (TdT) (1-6, 25-36, 41) have been used for the incor-poration of labeled nucleotides to DNA strand breaks in situ. The tailing reaction using TdT, which was also described as ISEL (in situ end labeling) (5, 35) or TUNEL (TdT-mediated dUTP nick end labeling) (1, 6, 31, 33) technique, has several advantages in comparison to the in situ nick translation (ISNT) using DNA polymerase:Label intensity of apoptotic cells is higher with TUNEL compared to ISNT, resulting in an increased sensitivity (2, 4). Kinetics of nucleotide incorporation is very rapid with TUNEL com-pared to the ISNT (2, 4).TUNEL preferentially labels apoptosis in comparison to necrosis, thereby discriminating apoptosis from necrosis and from primary DNA strand breaks induced by antitumor drugs or radiation (3, 4).2.2 Backgroundinformation,continued3. Procedures and required materialsThe working procedure described below has been developed andpublished by R. Sgonc and colleagues (6). The main advantage of thissimple and rapid procedure is the use of fluorescein-dUTP to labelDNA strand breaks. This allows the detection of DNA fragmentationby fluorescence microscopy directly after the TUNEL reaction priorto the addition of the secondary anti-fluorescein-POD-conjugate.3.1Flow chartAssay procedure The assay procedure is explained in the following flow chart.Adherent cells, cell smears and cytospin preparations Cryopreservedtissue sectionsParaffin-embeddedtissue sections↓↓↓Fixation ?Dewaxation ?Rehydration ?ProteasetreatmentPermeabilisation of samples↓Addition of TUNEL reaction mixtureOPTIONAL: Analysis of samples by fluorescence microscopy↓Addition of Converter-PODAddition of Substrate solution↓Analysis of samples by light microscopy3.2Preparation of sample material3.2.1Adherent cells, cell smears and cytospin preparationsAdditional solutions required ?Washing buffer: Phosphate buffered saline (PBS)Blocking solution: 3% H2O2 in methanolFixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-paredPermeabilisation solution: 0.1% Triton1) X-100 in 0.1% sodium citrate, freshly prepared (6)Procedure In the following table describes the fixation of cells, blocking of endo-genous peroxidase and cell permeabilisation.Note: Fix and permeabilisate two additional cell samples for the nega-tive and positive labeling controls.Step Action1Fix air dried cell samples with a freshly prepared Fixationsolution for 1 h at 15-25°C.2Rinse slides with PBS.3Incubate with Blocking solution for 10 min at 15-25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2-8°C).6Proceed as described under 3.3.3.2.2 Tissue sections3.2.2.1 Treatment of paraffin-embedded tissuePretreatment of paraffin embedded tissue Tissue sections can be pretreated in 4 different ways. If you use Pro-teinase K the concentration, incubation time and temperature have to be optimized for each type of tissue (1, 29, 33, 36, 40, 42).Note: Use Proteinase K only from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results!The other 3 alternative procedures are also described in the following table (step 2).Additional solutions required ?Xylene and ethanol (absolute, 95%, 90%, 80%, 70%, diluted in dou-ble distilled water) Washing buffer: PBSProteinase K, nuclease free (Cat. No. 745 723), working solution: [10-20 g/ml in 10 mM Tris/HCl, pH 7.4-8] Alternative treatmentsPermeabilisation solution: 0.1% Triton1) X–100, 0.1% sodium citrate, freshly preparedPepsin* (0.25% - 0.5% in HCl, pH 2) or trypsin*, 0.01 N HCl, nuclease free0.1 M Citrate buffer, pH 6 for the microwave irradiationProcedure In the following table the pretreatment of paraffin-embedded tissue with Proteinase K treatment and 3 alternative procedures aredescribed.Note: Add additional tissue sections for the negative and positivelabeling controls.Step Action1Dewax and rehydrate tissue section according to standardprotocols (e.g. by heating at 60°C followed by washing inxylene and rehydration through a graded series of ethanoland double dist. water) (1, 33, 36).2Incubate tissue section for 15-30 min at 21–37°C with Pro-teinase K working solution.Alternatives:Treatment:1. Permeabilisa-tion solutionIncubate slides for 8 min.2. Pepsin* (30, 40)or trypsin*15-60 min at 37°C.3. Microwave irradiation ?Place the slide(s) in a plastic jar containing 200 ml 0.1 M Citrate buffer, pH6.0.Apply 350 W microwave irradiation for 5 min.3Rinse slide(s) twice with PBS.4Proceed as described under 3.3.3.2.2.1 Treatment of paraffin-embedded tissue, continued3.2.2.2Treatment of cryopreserved tissueAdditional solutions required ?Fixation solution: 4% Paraformaldehyde in PBS, pH 7.4, freshly pre-paredWashing buffer: PBSBlocking solution: 3% H2O2 in methanolPermeabilisation solution (0.1% Triton1) X–100, 0.1% sodium citrate), freshly preparedCryopreserved tissue In the following table the pretreatment of cryopreserved tissue is described.Note: Fix and permeabilisate two additional samples for the negative and positive labeling controls.Step Action1Fix tissue section with Fixation solution for 20 min at 15–25°C.2Wash 30 min with PBS.Note:For storage, dehydrate fixed tissue sections 2 min inabsolute ethanol and store at ?15 to ?25°C.3Incubate with Blocking solution for 10 min at 15–25°C.4Rinse slides with PBS.5Incubate in Permeabilisation solution for 2 min on ice (2–8°C).6Proceed as described under 3.3.3.3Labeling protocol 3.3.1 Before you beginPreparation of TUNEL reaction mixture One pair of tubes (vial 1: Enzyme Solution, and vial 2: Label Solution) issufficient for staining 10 samples by using 50 ?l TUNEL reaction mix-ture per sample and 2 negative controls by using 50 ?l Label Solutionper control.Note : The TUNEL reaction mixture should be prepared immediately before use and should not be stored. Keep TUNEL reaction mixture onice until use.Additionalreagents required ?Micrococcal nuclease or ?DNase I, grade I (Cat. No. 104 132)Controls Two negative controls and a positive control should be included ineach experimental set up.StepAction 1Remove 100 ?l Label Solution (vial 2) for two negative con-trols.2Add total volume (50 ?l) of Enzyme solution (vial 1) to the remaining 450 ?l Label Solution in vial 2 to obtain 500 ?l TUNEL reaction mixture.3Mix well to equilibrate components.Negative control:Incubate fixed and permeabilized cells in 50 ?l/well Label Solution (without terminal transferase) instead of TUNEL reaction mixture.Positive control:Incubate fixed and permeabilized cells with micro-coccal nuclease or DNase I, grade I (3000 U/ml– 3 U/ml in 50 mM T ris-HCl, pH 7.5, 10 mM MgCl 2 1mg/ml BSA) for 10 min at 15-25°C to induce DNA strand breaks, prior to labeling procedures.3.3.2Labeling protocol for adherent cells, cell smears, cytospin preparations andtissuesAdditional equipment and solutions required ?Washing buffer: PBS ?Humidified chamber ?Parafilm or coverslip Procedure Please refer to the following table.Step Action1Rinse slides twice with PBS.2Dry area around sample.3Add50?l TUNEL reaction mixture on sample.Note: For the negative control add 50 ?l Label solution each.To ensure a homogeneous spread of TUNEL reaction mixtureacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or coverslip during incuba-tion.4Add lid and incubate for 60 min at 37°C in a humidified atmo-sphere in the dark.5Rinse slide 3 times with PBS.6Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wavelengthin the range of 450–500 nm and detection in the range of515–565 nm (green).3.3.3 Labeling protocol for difficult tissueAdditional equipment and solutions required ?Citrate buffer, 0.1 M, pH 6.0.Washing buffer: PBSTris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serumPlastic jarMicrowaveHumidified chamberProcedure Please refer to the following table.Step Action1Dewax paraformaldehyde- or formalin-fixed tissue sectionsaccording to standard procedures.2Place the slide(s) in a plastic jar containing 200 ml 0.1 MCitrate buffer, pH 6.0.3?Apply 750 W (high) microwave irradiation for 1 min.Cool rapidly by immediately adding 80 ml double dist.water (20–25°C).Transfer the slide(s) into PBS (20–25°C).DO NOT perform a proteinase K treatment!4Immerse the slide(s) for 30 min at 15–25°C in Tris-HCl, 0.1 MpH 7.5, containing 3% BSA and 20% normal bovineserum.5Rinse the slide(s) twice with PBS at 15–25°C.Let excess fluid drain off.6Add50µl of TUNEL reaction mixture on the section and.Note: For the negative control add 50 µl Label solution.7Incubate for 60 min at 37°C in a humidified atmosphere in thedark.8?Rinse slide(s) three times in PBS for 5 min each.Samples can be analyzed in a drop of PBS under a fluores-cence microscope at this state. Use an excitation wave-length in the range of 450–500 nm and detection in therange of 515–565 nm (green).3.4 SignalconversionAdditional equipment and solutions required ?Washing buffer: PBSHumidified chamberParafilm or coverslipDAB Substrate* (Cat. No. 1 718 096) or alternative POD substrate Mounting medium for light microscopy Procedure Please refer to the following table.Step Action1Dry area around sample.2Add50?l Converter-POD (vial 3) on sample.Note: To ensure a homogeneous spread of Converter-PODacross cell monolayer and to avoid evaporative loss, samplesshould be covered with parafilm or cover slip during incuba-tion.3Incubate slide in a humidified chamber for 30 min at 37°C.4Rinse slide 3× with PBS.5Add 50–100 ?l DAB Substrate or alternative POD substrates.6Incubate slide for 10 min at 15–25°C.7Rinse slide 3× with PBS.8Mount under glass coverslip (e.g. with PBS/glycerol) and ana-lyze under light microscope.Alternative: Samples can be counterstained prior to analysisby light microscope.4. Appendix4.1 Trouble-shootingThis table describes various troubleshooting parameters. Problem Step/Reagent of ProcedurePossible cause RecommendationNonspecific labeling Embedding of tissue UV-irradiation forpolymerization ofembedding material(e.g. methacrylate)leads to DNA strandbreaksTry different embedding materialor different polymerizationreagent.Fixation Acidic fixatives (e.g.methacarn, Carnoy’sfixative)Try 4% buffered paraformal-dehyde.Try formalin or glutaralde-hyde.TUNEL reaction TdT concentration toohighReduce concentration of TdT bydiluting it 1:2 up to 1:10 withTUNEL Dilution Buffer (Cat. No.1 966 06).Converter solution Endogenous PODactivityBlock endogenous POD byimmersing for 10 min in 3%H2O2 in methanol prior to cellpermeabilisation.Non-specific bindingof anti-fluorescein-PODBlock with normal anti-sheepserum.Block for 20 min with PBScontaining 3% BSA.Reduce concentration ofconverter solution to 50%. Nucleases Some tissues (e.g. smooth muscles)show DNA strandbreaks very soon aftertissue preparationFix tissue immediately afterorgan preparation.Perfuse fixative through livervein.Some enzymes arestill activeBlock with a solution containingddUTP and dATP.continued on next page。
翻译好的罗氏公司Tunel试剂盒操作说明方案
罗氏(R o c h e)公司T u n e l试剂盒操作说明书(Insitucelldeathdetectionkit-POD法)一、原理：TUNEL（TdT-mediateddUTPnickendlabeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdTEnzyme）的作用下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radishperoxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含：1号（蓝盖）EnzymeSolution酶溶液：TdT10×、2号（紫盖）LabelSolution标记液：荧光素标记的dUTP1×、3号（棕瓶）Converter-POD:标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5μl20×DAB+1μL30%H2O2+94μlPBS）、ProteinaseK工作液（10-20μg/mlin10mMTris/HCl，pH7.4-8）或细胞通透液（0.1%TritonX-100溶于0.1%柠檬酸钠，临用前配制）、苏木素或甲基绿、DNase1（3000U/ml–3U/mlin50mMTris-HCl，pH7.5，10mMMgCl2，1mg/mlBSA）等。
TUNEL染色
相关疾病：•产品名称：罗氏(Roche)公司Tunel试剂盒英文名称：Tunel产品货号：产品规格：50T试剂级别：试剂级产品产地：USA产品商标：RocheIn situ cell death detection kit-POD法一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡初期进程中细胞核DNA的断裂情形。
其原理是荧光素（fluorescein）标记的dUTP 在脱氧核糖核苷酸结尾转移酶（TdT Enzyme）的作用下，能够连接到凋亡细胞中断裂DNA的3’-OH结尾，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者又与HRP底物二氨基联苯胺（DAB）反映产生很强的颜色反映（呈深棕色），特异准确地定位正在凋亡的细胞，因此在光学显微镜下即可观看凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因此没有3‘-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评判，和通过双色法确信细胞死亡类型和分化时期。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含TdT 10×、荧光素标记的dUTP 1×、标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5 μl 20×DAB+1μL 30%H2O2+94 μl PBS）、ProteinaseK工作液（10-20 μg/ml in 10 mM Tris/HCl，pH ）或细胞通透液（% Triton X-100 in % sodium citrate，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl，pH ，10 mM MgCl2，1 mg/ml BSA）等。
【求助】组织TUNEL染色
【求助】组织TUNEL染⾊产品名称：罗⽒(Roche)公司Tunel试剂盒英⽂名称： Tunel产品货号： 11684817910产品规格： 50T试剂级别：试剂级产品产地： USA产品商标： RocheIn situ cell death detection kit-POD法⼀、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是⽤来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是荧光素（fluorescein）标记的dUTP在脱氧核糖核苷酸末端转移酶（TdT Enzyme）的作⽤下，可以连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，后者⼜与HRP底物⼆氨基联苯胺（DAB）反应产⽣很强的颜⾊反应（呈深棕⾊），特异准确地定位正在凋亡的细胞，因⽽在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞⼏乎没有DNA断裂，因⽽没有3‘-OH形成，很少能够被染⾊。
本试剂盒适⽤于组织样本（⽯蜡包埋、冰冻和超薄切⽚）和细胞样本（细胞涂⽚）在单细胞⽔平上的凋亡原位检测。
还可应⽤于抗肿瘤药的药效评价，以及通过双⾊法确定细胞死亡类型和分化阶段。
⼆、器材与试剂器材：光学显微镜及其成像系统、⼩型染⾊缸、湿盒（塑料饭盒与纱布）、塑料盖玻⽚或封⼝膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含TdT 10×、荧光素标记的dUTP 1×、标记荧光素抗体的HRP；⾃备试剂：PBS、双蒸⽔、⼆甲苯、梯度⼄醇（100、95、90、80、70%）、DAB⼯作液（临⽤前配制，5 µl 20×DAB+1µL 30%H2O2+94 µl PBS）、Proteinase K⼯作液（10-20 µg/ml in 10 mM Tris/HCl， pH 7.4-8）或细胞通透液（0.1% Triton X-100 in 0.1% sodium citrate，临⽤前配制）、苏⽊素或甲基绿、DNase1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl，pH 7.5， 10 mM MgCl2，1 mg/ml BSA）等。
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In situ cell death detection kit-POD法一、原理：TUNEL（TdT-mediated dUTP nick end labeling）细胞凋亡检测试剂盒是用来检测组织细胞在凋亡早期过程中细胞核DNA的断裂情况。
其原理是脱氧核糖核苷酸末端转移酶（TdT Enzyme）把荧光素（fluorescein）标记的dUTP（三磷酸脱氧尿甘）连接到凋亡细胞中断裂DNA的3’-OH末端，并与连接辣根过氧化酶（HRP，horse-radish peroxidase）的荧光素抗体特异性结合，HRP又与HRP底物二氨基联苯胺（DAB）反应产生很强的颜色反应（呈深棕色），特异准确地定位正在凋亡的细胞，因而在光学显微镜下即可观察凋亡细胞；由于正常的或正在增殖的细胞几乎没有DNA断裂，因而没有3‘-OH形成，很少能够被染色。
本试剂盒适用于组织样本（石蜡包埋、冰冻和超薄切片）和细胞样本（细胞涂片）在单细胞水平上的凋亡原位检测。
还可应用于抗肿瘤药的药效评价，以及通过双色法确定细胞死亡类型和分化阶段。
二、器材与试剂器材：光学显微镜及其成像系统、小型染色缸、湿盒（塑料饭盒与纱布）、塑料盖玻片或封口膜、吸管、各种规格的加样器及枪头等；试剂：试剂盒含TdT 10×、荧光素标记的dUTP 1×、标记荧光素抗体的HRP；自备试剂：PBS、双蒸水、二甲苯、梯度乙醇（100、95、90、80、70%）、DAB工作液（临用前配制，5 μl 20×DAB+1μL 30%H2O2+94 μl PBS）、Proteinase K工作液（10-20 μg/ml in 10 mM Tris/HCl， pH 7.4-8）或细胞通透液（0.1% Triton X-100 in 0.1% sodium citrate，临用前配制）、苏木素或甲基绿、DNase 1（3000 U/ml– 3 U/ml in 50 mM Tris-HCl，pH 7.5， 10 mM MgCl2，1 mg/ml BSA）等。
三、实验步骤操作流程图：制作石蜡切片→脱蜡、水合→细胞通透→加TUNEL反应液→加converter-POD→与底物DAB反应显色→光学显微镜计数并拍照。
具体操作步骤（石蜡包埋切片的检测）：1. 用二甲苯浸洗2次，每次5min；2. 用梯度乙醇（100、95、90、80、70%）各浸洗1次，每次3min；注：上面两步是针对石蜡切片样本的处理4. 用Proteinase K工作液处理组织15-30 min 在21–37°C（温度、时间、浓度均需摸索）或者加细胞通透液8min；5. PBS漂洗2次；6. 制备TUNEL反应混合液，处理组用50μl TdT+450μl 荧光素标记的dUTP液混匀；而阴性对照组仅加50μl 荧光素标记的dUTP液，阳性对照组先加入100μl DNase 1，反应在15~25℃×10min，后面步骤同处理组。
7. 玻片干后，加50μl TUNEL反应混合液（阴性对照组仅加50μl 荧光素标记的dUTP液）于标本上，加盖玻片或封口膜在暗湿盒中反应37℃×1h。
8. PBS漂洗3次；9. 可以加1滴PBS在荧光显微镜下计数凋亡细胞（激发光波长为450~500nm，检测波长为515~565nm）；10. 玻片干后加50μl converter-POD于标本上，加盖玻片或封口膜在暗湿盒中反应37℃×30min。
11. PBS漂洗3次；12. 在组织处加50~100μlDAB底物，反应15~25℃×10min；13. PBS漂洗3次；14. 拍照后再用苏木素或甲基绿复染，几秒后立即用自来水冲洗。
梯度酒精脱水、二甲苯透明、中性树胶封片。
15. 加一滴PBS或甘油在视野下，用光学显微镜观察凋亡细胞（共计200~500个细胞）并拍照。
可结合凋亡细胞形态特征来综合判断（未染色细胞变小，胞膜完整但出现发泡现象，晚期出现凋亡小体，贴壁细胞出现邹缩、变圆、脱落；而染色细胞呈现染色质浓缩、边缘化，核膜裂解，染色质分割成块状/凋亡小体）对于培养细胞的预处理：①在载玻片上铺一层薄薄的多聚赖氨酸（见备注4），干燥后在去离子水中漂洗，干燥后4℃保存；②适当方法诱导细胞凋亡，同时设未经诱导的对照组，各组离心收集约1×106个细胞，PBS洗一次，重悬，加到铺好的多聚赖氨酸载玻片上，自然干燥，使细胞很好的吸附到载玻片上；③将吸附细胞的载玻片在4%多聚甲醛（见备注2）中固定25min；④PBS浸洗二次，每次5min；⑤将吸附细胞的载玻片在0.2%的Triton X-100（见备注5）中处理5min；⑥PBS浸洗二次，每次5min；后续操作如同石蜡包埋切片的6—15四、注意事项1. 进行PBS 清洗时，每次清洗5 min。
2. PBS清洗后，为了各种反应的有效进行，请尽量除去PBS 溶液后再进行下一步反应。
3. 在载玻片上的样本上加上实验用反应液后，请盖上盖玻片或保鲜膜，或在湿盒中进行，这样可以使反应液均匀分布于样本整体，又可以防止反应液干燥造成实验失败。
4. TUNEL反应液临用前配制，短时间在冰上保存。
不宜长期保存，长期保存会导酶活性的失活。
5. 如果20×DAB 溶液颜色变深成为紫色，则不可使用，需重新配制。
6. 用甲基绿（Methyl Green）染液（3-5%甲基绿溶于0.1M 醋酸巴比妥 PH4.0）染色后，请用灭菌蒸馏水清洗多余的甲基绿。
然后进行洗净（100%乙醇）、脱水（二甲苯）透明、封片后通过光学显微镜观察操作。
如果此时使用80~90%的乙醇洗净时，甲基绿比较容易脱色，注意快速进行脱水操作。
7. 荧光素标记的dUTP液含甲次*酸盐和二氯钴等致癌物，可通过吸入、口服等途径进入机体，注意防护。
8. 试剂保存；未打开的试剂盒贮存在-20℃（-15~25℃）；converter -POD液一旦解冻，以后就保存在4℃（2~8℃）下，至少在6 m内稳定，避免再次冻存；TUNEL反应液临用前配好后，放至冰上直至使用。
9. 结果分析时注意：在坏死的晚期阶段或在高度增殖/代谢的组织细胞中可产生大量DN**断，从而引起假阳性结果；而有些类型的凋亡性细胞死亡缺乏DNA断裂或DNA裂解不完全，以及细胞外的矩阵成分阻止TdT进入胞内反应，进而产生假阴性结果。
newfish的实验步骤大概如下：1、石蜡切片脱蜡至水，2、0.3%H2O2的甲醇液室温作用5分钟，0.01mol/lPBS清洗两次，每次5分钟3、20ug/ml蛋白酶K处理，湿盒内37℃孵育30min，PBS清洗，4、加入TUNEL反应混合液50ul,盖上蜡膜，湿盒内37℃孵育1小时，PBS清洗，5、3%BSA室温作用20min,PBS清洗6、POD转换液50ul,盖上蜡膜，湿盒内37℃孵育30min，PBS清洗7、DAB显色5~10min，自来水冲洗，8、苏木素复染，盐酸酒精分化20s，自来水冲洗20min，9、梯度脱水50%，70%，80%，90%，100%（1），100%（2），2min/次二甲苯透明两次，10min/次，中性树胶封固，镜下观察。
TUNEL法检测细胞凋亡实验原理和方法细胞凋亡中染色体DNA的断裂是个渐进的分阶段的过程，染色体DNA首先在内源性的核酸水解酶的作用下降解为50-300kb的大片段。
然后大约30 ﹪的染色体DNA在Ca ²+和Mg²+依赖的核酸内切酶作用下，在核小体单位之间被随机切断，形成180～200bp核小体DNA多聚体。
DNA双链断裂或只要一条链上出现缺口而产生的一系列DNA的3’-OH末端可在脱氧核糖核苷酸末端转移酶（TdT）的作用下，将脱氧核糖核苷酸和荧光素、过氧化物酶、碱性磷酸化酶或生物素形成的衍生物标记到DNA的3’-末端，从而可进行凋亡细胞的检测，这类方法一般称为脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（TUNEL）。
由于正常的或正在增殖的细胞几乎没有DNA的断裂，因而没有3’-OH形成，很少能够被染色。
低分子量的DNA分离后，也可使用DNA聚合酶进行缺口翻译（nick translation），使低分子量的DNA标记或染色，然后分析凋亡细胞。
TUNEL或缺口翻译法实际上是分子生物学与形态学相结合的研究方法，对完整的单个凋亡细胞核或凋亡小体进行原位染色，能准确的反应细胞凋亡最典型的生物化学和形态特征，可用于石蜡包埋组织切片、冰冻组织切片、培养的细胞和从组织中分离的细胞的细胞凋亡测定，并可检测出极少量的凋亡细胞，灵敏度远比一般的组织化学和生物化学测定法要高，因而在细胞凋亡的研究中已被广泛采用。
一、过氧化物酶标记测定法原理：脱氧核糖核苷酸衍生物地高辛[（digoxigenin）-11-dUTP]在TdT酶的作用下，可以掺入到凋亡细胞双链或单链DNA的3-OH末端，与dATP形成异多聚体，并可与连接了报告酶（过氧化物酶或碱性磷酸酶）的抗地高辛抗体结合。
在适合底物存在下，过氧化物酶可产生很强的颜色反应，特异准确的定位出正在凋亡的细胞，因而可在普通光学显微镜下进行观察。
毛地黄植物是地高辛的唯一来源。
在所有动物组织中几乎不存在能与抗地高辛杭体结合的配体，因而非特异性反应很低。
抗地高辛的特异性抗体与脊椎动物甾体激素的交叉反应不到1％，若此抗体的Fc部分通过蛋白酶水解的方法除去后，则可完全排除细胞Fc 受体非特异性的吸附作用。
本方法可以用于福尔马林固定的石蜡包埋的组织切片、冰冻切片和培养的或从组织中分离的细胞凋亡测定。
检测晚期凋亡。
基本原理：对不同组织切片先增加细胞膜通透性，然后让rTDT和bio 标记的dTUTP进入细胞内，在rTDT的辅助下dTUTP与核断裂的DNA 3’-OH结合，再用HRP标记的链霉亲和素与dTUTP上biotin结合（每个链霉亲和素至少可以再结合3个biotin分子），最后用DAB、过氧化氢与SP上的辣根过氧化物酶HRP发生氧化、环化反应，形成苯乙肼聚合物而呈现棕褐色，最终通过计数每张切片上不同视野中TUNEL阳性细胞的比例来判断细胞凋亡发生情况。
罗氏试剂盒一、试剂1、酶浓缩溶液5×50μl——末端脱氧核糖核酸转移酶（10×）二、试剂2、标记溶液5×550μl——含有核苷酸混合液的反应液（1×）三、试剂3、转化剂-POD 3.5ml——酶标记抗荧光素抗体（即用型，融后4℃保存）四、二、所需的溶液和仪器10mM Tris/HCl（pH7.4~7.8）、PK配制、DAB、饭盒、吹风机、3%H2O2甲醇溶液、PBS、盖玻片、中性树胶、苏木素、二甲苯和无水乙醇（用量多）、双蒸水（用以配梯度酒精）①充分脱蜡和水化。