For whole-mount TUNEL (terminal deoxynucleotidyl transfera-mediated dUTP nick and labeling) staining, roots were fixed overnight in 4% (v/v) paraformaldehyde in PBS
at room temperature. Fixed root tissues were washed five times with PBS, treated with Proteina K (20 _g ml_1, Invitrogen) in 10 mM Tris-HCl (pH 7.5) at 37 °C for 30 min, and then washed three times with PBS. TUNEL reaction was performed in a microcentrifuge tube (1.5 ml) using the In Situ cell death detection
kit with fluorescein (Roche Applied Science) according to the manufacturer’s instructions. To visualize nuclei in root cells, samples were stained using
4_,6-diamidino-2-phenylindo le (DAPI, Sigma-Aldrich) at 0.5 _g ml_1 in 0.1%(v/v) Triton X-100 for 10 min, then washed twice with water. DAPI-stained
and TUNEL-positive nuclei were obrved under a Leica MZ
面试礼仪培训FLIII stereo fluorescence microscope equipped with a BFP filter
t (excitation 390/32 nm, emission 460/50 nm, dichroic 420
nm) and an enhanced green fluorescence protein filter t,
respectively
E, microscopic analysis of TM-treated (_) and untreated (_) root cells.
The cells were counterstained in situ with DAPI followed by TUNEL reagents. Corresponding pha contrast image (PhC) of root cells is also shown. Bars_100_m.
To determine
endonuclea activity, the DeadEnd Fluor imetric TUNEL System (Promega) was
ud. The technique was adjusted for the study of the whole root and obrvation of
epidermal cells. After treatment, whole roots were fixed with paraformaldehyde and
pasted using inert silicon adhesive to the surface of a micr oscope slide (roots were
not sliced). Cell walls were per meabilid for 10 minutes using cellulolytic and
pectolytic enzymes (1.5% cellula Onozuka RS, 1% cellulysin, 0.1% pectolya Y-
23, 0.1 mM KCl, 0.1 mM C aCl2, pH 6.0 adjusted by 4 mM MES/2 mM Tr is).
TUNEL a ssay1
To detect cell apoptosis caud by PEG stress, an in situ apoptosis detection kit (Takara Bio Inc., Shiga, Japan) was ud. The TUNEL assay was performed according to the manufacturer’s instructions with a few modifications. In brief, the whole edlings were fixed in 4% paraformaldehyde ⁄PBS (pH 7.4) solution at 4_C overnight. After being washed with 1·PBS solution three times, the samples were immerd into 70% ethanol for at least 24 h at )20_C. After the samples were washed three times with PBS, they were subjected to a permeabilization buffer for 15 min on ice and followed by a PBS wash. Then the samples were transferred into 50 ll Reaction Buffe
r (TdT Enzyme 5 ll + Labeling Safe Buffer 45 ll) for a 90-min incubation at 37_C. The labeling procedure was stopped by washing with a PBS solution. The image was acquired with a Zeiss LSM 510 Confocal lar scanning microscope with a 488 nm excitation line and a 530 nm emission filter.
TUNEL assay
For the TUNEL assay, 7-day-old dark-grown edling roots were fixed and prepared in the same manner as for immunostaining. Following tissue permeabilization, the TUNEL reaction was performed using the In Situ Cell Death Detection Kit Fluorescein (Roche, Mannheim, Germany). For the positive control, wild-type roots were incubated for 10 minutes with 20U DNa I (Sigma-Aldrich) in 50 mM Tris-HCl pH 7.5, 1 mg/ml BSA, 1 mM MgSO4 and subquently washed three times with PBS buffer prior to the TUNEL reaction. For the negative control, label solution lacking the enzyme was added. For DAPI staining, slides were incubated with 1 μg/ml DAPI for 30 minutes, washed three times with PBS and embedded with antifade [PBS containing 90% glycerol and 25一封家书500字
mg/ml 1,4-diazabicyclo(2.2.2)octane, pH 9.5]. Fluorescence microscopy was performed with a Leica Confocal SP2 (Leica, Heidelberg, Germany).
TUNEL assay2
Paraffin ctions (8-lm thick) were subjected to TUNEL reaction with an in situ Apoptosis Detection kit (Takara Bio). Samples were obrved under a confocal microscanning lar microscope (FV10; O
lympus).
Illustrated in Figure 2(a) is the fluorescence emission profile using the DAPI-FITC filter block and a culture of Indian Muntjac deerskin fibroblast cells stained with Alexa Fluor 488 conjugated to phalloidin, which binds to the intracellular filamentous actin network. The visible light absorption maximum of Alexa Fluor 488 is 495 nanometers and the emission maximum occurs at 519 nanometers in the green region of the spectrum. In addition, the specimen was simultaneously stained with DAPI (targeting DNA in the cell nucleus; excitation at 358 nanometers and emission at 461 nanometers) and MitoTracker Red CMXRos (targeting mitochondria; red emission). Note the ab
nce of signal from the red fluorophore (MitoTracker), but the prence of bright green fluorescence exhibited by the actin filaments and the slightly lower intensity blue signal from DAPI in the cell nucleus.
理论上讲，你配制0.1%柠檬酸钠100ml，然后加入trito n 0.1 ml 即可，两种物质的浓度都是0.1%, 不同的只是一个是质量比一个是体积比。你可以问问试剂公司的技术部具体怎么配。
作TUNEL用0.1%TritonX-100是为了增加细胞膜的通透性，使后续的酶和核苷酸能进入细胞核内（DNA断端延续），所以使用时需控制时间，不能过度使用。
应该是先配制枸橼酸-枸橼酸钠缓冲液，然后用此缓冲液配制0.1%的TritonX-100。
(应该是先配制枸橼酸-枸橼酸钠缓冲液，然后用此缓冲液配制0.1%的TritonX-100。)
我看书上是这样配的：柠檬酸0.4g
柠檬酸三钠 3.0g
加双蒸水至1000ml 。
因为没配过，还请赐教，谢谢！！！
阴虚火旺证
柠檬酸0.4g
柠檬酸三钠 3.0g
加双蒸水至1000ml 。
就是枸橼酸-枸橼酸钠缓冲液（枸橼酸又叫柠檬酸），用此液再去配制0.1%的Trito nX-100（1ml缓冲液中加1ul TritonX-100）。
柠檬酸-柠檬酸钠缓冲液（0.1 mol/L）pH 4.2
0.1 mol/L柠檬酸(mL) 12.3 +0.1 mol/L柠檬酸钠(mL)7.7
柠檬酸：C6H8O7•H2O分子量＝210.14 ；0.1 mol/L溶液为21.01 g/L。
柠檬酸钠：Na3C6H5O7•2H2O分子量＝294.12 ；0.1 mol/L溶液为29.41 g/L。
称取固体40g，加入上述PBS液500ml中，加热至50-60度，不断搅拌，搅拌同时缓慢加入NaOH直至溶解澄清，然后加PBS液至1000ml.
1。1000ml (0.1M)柠檬酸钠溶液配制
精确称取29.41g柠檬酸钠（用折好的硫酸纸），投入200ml烧杯中用适量蒸馏水溶解，溶解液转移到1000ml的容量瓶，加入100%乙醇10ml ，用蒸馏水定容到1000ml。转移到干净试剂瓶中，贴标备用。
A液：0.1M柠檬酸21克柠檬酸溶于1000ML水，高压消毒
B液：0.1M柠檬酸钠29.4克柠檬酸钠溶于1000ML水高压消毒
修复缓冲液：
450ML水再加入以下成份：
0.1M柠檬酸9.5ML
0.1M柠檬酸钠40.5ML
枸橼酸就是柠檬酸
国学经典语录2.0.1%枸橼酸钠溶液是0.1g溶于100ml蒸馏水中吗？
PBS漂洗所用的浓度是0.05M（PH7.6）；在文献中看到用的Triton-100多是1％的，若你是要配0.1％
坐火车提前多久进站
的Triton-100，0.1％的柠檬酸钠当然就是在1L水里面加入1g柠檬酸钠固体了。
针对问题2（TU NEL法的实验原理是什么？）：
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dTU TP进入细胞内，在rTDT的辅助下dTU TP与核断裂的DNA 3’-O H结合，再用HRP标记的链霉亲和素与dTU TP上的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DA B、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TU NEL阳性细胞的比例来判断细胞凋亡发生情况。
针对问题3（TU NEL实验中几个关键步骤是什么？）：
2. 把握好细胞通透的时间。一般根据切片的厚薄，选择蛋白酶k的孵育时间，常用10~30min，几um切片用短时间；几十um切片用长时间，通过摸索达到既不脱片，有能够使后面的酶和抗体进入胞内。
3. 适当延长TU NEL反应液的时间。一般是37度1h，你也可以根据你的凋亡损伤程度，选择更长的时间，可长至2h，但要结合你最终的背景着色。
5.PBS的充分清洗。我个人认为，在TU NEL反应后和酶标反应后的清洗应十分严格，可增加次数达5次，因为这些清洗直接决定最后切片的非特异性着色。
正对照：加100μL DNa Ⅰ缓冲液室温下孵育5min 。轻拍去水分，加100μL 含有
5.5-10units/mL 的DNa Ⅰ的酶缓冲液，室温下孵育10min。去除多余水分，在充足的去
礼品推销
离子水中洗涤3-4次。按照步骤1-12处理正对照切片。
注：DNaⅠ处理细胞会使染色体DNA片段化，并暴露出大量的可供标记的3’-OH。
四言对联
应当使用另外一个染色缸处理正对照切片，因为残留的DNa Ⅰ的活性会给实验组引入强烈的背景。
DAPI Working Solution 0.1 µg/ml DAPI in Assay Buffer (Prepared from a 1 mg/ml stock solution; e previous
4％多聚甲醛配制方法
1) 900 ml dd H20 + 500 ul 1N NaOH
2) Heat to 65-70℃
3) While stirring, add 40 g parafo rmaldehy de
4) C ontinue heating and stirring until parafo rmaldehy de is dissolv ed
5) Let cool to room temperature
6) A dd 100 ml of 10X PBS
7) pH to 7.3 w ith HC l
8) Sterile w ith f ilter
9) Store at 4℃, protected f rom light
加热时注意不要使之沸腾，因为需要现用现配所以我一般就配10毫升。
用一个带盖的玻璃离心管加0.4克多聚甲醛，加8毫升PBS，加1毫升2N的NAOH，放到60度的水浴里，10分钟，拿出来用手颠倒几次，基本上就都溶了，再用HCL调ph到7.2，最后定容就行了，我最快10分钟搞定。注意：1.配制时应在通风条件下操作，并避免接触皮肤及吸入（戴口罩及手套），因PFA有较强的毒性，对粘膜及皮肤有刺激作用。2.加热时，温度不可过高，常为60－65℃。否则PFA降解失效，配制好的PFA虽可有效一点时间，但过久的液体，固定效果下降，应用前新鲜配制。多聚甲醛室温不易溶解，加碱、加热可助溶，加热不必一定60度，不煮沸蒸发就行，加碱后需要调回PH值。加碱后调回PH值不影响使用待冷却至室温调PH值（配好后一周之内使用，也不一定非要放四度保存的，室温也能行），
PBS用0.1mol/L
一般实验不需要过滤及无酶水（DEPC水）
做免疫荧光最好新鲜配制，一次少配些，配多了放在4度也可以用一段时间
至于DAPI和TUNEL，放心不会有反应的
不钮扣的女孩DAPI插入双链DNA，而TU NEL只是和断裂的DNA末端链接上，再进行酶促反应，二者再空间上还是有很大距离的
在加抗体或TU NEL等贵重试剂时候，我们实验室一般是将反应液加到玻片上（25ul/样品），然后将玻片盖过去，待反应完成后，在盖子上加过量的PBS浸没玻片，后小心将玻片用镊子取出，这样可以省一半左右的试剂，而且结果不比用在玻片上面加大量的反应液的效果差。
1 TUNEL的绿色荧光淬灭非常快，我的大约5分钟就淬灭了，没来得及照满意的照片。而DAPI几个小时都还很明显。不知道大家的是不是这
样？
2 用抗淬灭封片剂会有用吗？如何解决这个问题
3 是不是DAPI染色重会加重TU NEL的淬灭？
1 当然要用抗淬灭封片剂F luorescence在普通光照10分钟就会严重淬灭。另外还要特别注
意避光操作。
2 不清楚DAPI染色会不会促进TU NEL的淬灭，据我所知，碘化丙啶可以接受fluorescein激发
产生的荧光，从而起到淬灭作用。可以查查荧光激发吸收图。
dapi的作用是复染细胞核
第四个问题，如果你的试剂盒里有直接封片染DAPI的话那就简单了一步，我没用过。我是在终止反应洗完，用1:1000P BS稀释DAP I室温避光孵育30分钟，然后再洗三次封片，效果也不错。最终目的都是染核，因为TU NEL检测的是断裂的DNA的3‘-O H，在细胞核内，如果二
者merage一下就可以看出凋亡的存在及比例。
TUNEL细胞凋亡检测程序
一、原理与意义：
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，其原理是生物素biotinylate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，hor-radish peroxida）的链霉亲和素streptavidin特异性结合，后者又与POD底物H2O2、二氨基联苯胺（DAB）产生深棕色反应，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
1. 器材：光学显微镜及其成像系统、摇床、组化笔、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、各种规格的移液器及枪头等。
2. 试剂：试剂盒含TdT 2×、Biotinylated标记的dUTP、标记streptavidin的HRP、SSC 20×、Proteina k、DAB 20×、DAB底物Buffer 20×、H2O2 20×；
3. 自备试剂：
1) 1×PBS（pH 7.4，2.5L＝20.0157g137mMNaCl＋0.499552g2.68mMKCl＋0.50013075g1.47mMKH2PO4＋
7.253337g8.1mMNa2HPO4），115℃×15 min；
2) 0.85％NaCl 500 ml＝4.25 gNacl＋500 ml三蒸水，115 ℃×15 min；
3) 4％多聚甲醛500 ml＝20 g多聚甲醛＋500 ml PBS在65 ℃水箱中3 h多，再放入4 ℃保存。
4) DNa I buffer：40 mMTris-HCl (pH 7.9)＋10 mMNaCl＋6 mMMgCl2＋10 mMCaCl2；
5) Proteina K buffer：100 mMTris-HCl (pH 8.0)＋50 mMEDTA；
