| Product Type | Immunohistochemistry Kit |
| Units | 60 Tests |
| Application | Immunohistochemistry |
BackgroundCell death occurs by two major mechanisms, necrosis and apoptosis. Apoptosis is also known as programmed cell death or ankoikis (a form of apoptosis which is induced by anchorage-dependent cells detaching from the surrounding extracellular matrix). Apoptosis leads to the elimination of cells without releasing harmful substances into the surrounding area. Too little or too much apoptosis plays a role in a great many diseases. When apoptosis functions inappropriately, cells that should be eliminated survive and potentially become immortal, as in cancer or leukemia. When apoptosis works overly well, too many cells may ‘die’ and the result may be grave tissue damage. This is the case in stroke and neurodegenerative disorders such as Alzheimer, Huntington and Parkinson diseases. The term ‘apoptosis’ refers only to the structural changes a cell goes through during the process of programmed cell death and not to the process itself. Classical necrotic cell death occurs due to noxious injury or trauma to the cell while apoptosis is an energy dependent mechanism that takes place during normal cell development. While necrotic cell death results in cell lysis, cellular apoptosis is characterized morphologically by cell shrinkage, nuclear pyknosis, chromatin condensation, and blebbing of the plasma membrane. Apoptosis is the result of a cascade of molecular and biochemical events involving endogenous endonucleases that cleave DNA into the prototypical ‘ladder of DNA fragments’ that may be visualized in agarose gels. Observation of oligonucleosomal DNA fragments by DNA laddering has long been the most acceptable and only available assay for the detection of apoptosis. Exalpha’s DNA Fragmentation Detection Kit exploits the fact that apoptotic endonucleases not only affect cellular DNA by producing the classical DNA ladder but also generate free 3’-OH groups at the ends of these DNA fragments. These free 3’-OH groups are end-labeled by the DNA Fragmentation Detection Kit allowing for the detection of apoptotic cells using a molecular biology-based, end labeling technique.
ApplicationsOptimal concentration should be evaluated by serial dilutions.
StorageExalpha’s DNA Fragmentation Detection Kit components are shipped on cold pack. Upon receipt, store kit at -20°C in a non-frost-free freezer. For long term storage, it is recommended that you aliquot and freeze the TdT Enzyme (Component 4), TdT Labeling Reaction Mix (Component 3), and 25x Streptavidin-HRP Conjugate (Component 7) at -20 °C. Thirty (30) minutes prior to use of each component, thaw component and keep on cold block or on ice. Return the components to -20°C for long term storage or 4-8ºC for short term storage (up to 2 weeks) immediately after use. Special care should be taken to keep TdT Enzyme (Component 4), TdT Labeling Reaction Mix (Component 3) and 25x Streptavidin-HRP Conjugate (Component 7) cold by pulling out the number of aliquots needed for the test, keeping them on ice, and leaving the remaining aliquots at -20°C.
CautionThis product is intended FOR RESEARCH USE ONLY, and FOR TESTS IN VITRO, not for use in diagnostic or therapeutic procedures involving humans or animals. It may contain hazardous ingredients. Please refer to the Safety Data Sheets (SDS) for additional information and proper handling procedures. Dispose product remainders according to local regulations.This datasheet is as accurate as reasonably achievable, but Nordic-MUbio accepts no liability for any inaccuracies or omissions in this information.
Kit ManualClick here to download the Kit Manual Safety Datasheet(s) for this product:NM_X2044K SDS_V1/wp-content/uploads/SDS/X2044K SDS_V1.pdf
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其次,察看次目的蛋白的存在形式,有没有多聚体形式及变构形式;
最后,查看多家抗体公司的DATA,看看别人的WB做出来的条带的位置。
根据你说的,特异识别多个组织中的同样大小的条带,我觉得很可能就是你的目的蛋白。
一般来说多克隆的阳性率高一些,但出现假阳性的比例也高一些。
1取组织
(1) 灌注固定:暴露心脏,将注射针头插入左心室,剪开右心耳,快速灌注PBS,待右心耳处流出澄清液体,换4%多聚甲醛灌注到大鼠四肢强直为止,快速取脑和肾。
(2) 固定:将组织置于4%多聚甲醛中固定15小时
(3) 脱水:将组织放入30% 蔗糖溶液中直至下沉
(4) 切片:脑组织冰冻切片20微米,37度烘干2h以上;
(5)将切片放置-80度保存,随用随取。
2免疫单标记
(1)做抗原修复(0.4g柠檬酸、3g柠檬酸钠、1000ml水配制成溶液,放到水浴箱中90~94℃保存15min),冷却后, 用1*PBS反复洗标本10min,吸去PBS,同时向标本加渗透液(配0.4%trition液,然后将BSA加入到配好的trition液中,配成1%浓度)100ul,常温下湿盒反应1h。
(2) 吸去渗透液,同时向标本加block液(5%封闭用正常山羊血清)100ul,常温下湿盒反应5h。
(3) 吸去block液,同时向标本加入1:50稀释的羊抗鼠一抗100ul,37度湿盒反应过夜。
(4)吸去一抗,并回收,用1*PBS洗液反复洗15min。
(5)快速从冰箱中取出驴抗羊的二抗,并立即用锡纸包裹,注入到一个新的以锡纸包裹的tube管中并用PBS以1:200稀释。
(6)进入暗室,吸去PBS,同时加入二抗100ul,37度湿盒反应2h,封片。
(7)放置到荧光显微镜下观察。
又由于自然存在的抗原大都存在多个抗原表位,会刺激机体产生多种针对同一抗原的不同抗原表位相应的不同抗体.

