Ki67antigenistheprototypiccellcyclerelatednuclearprotein,expressedbyproliferatingcellsinallphasesoftheactivecellcycle(G1,S,G2andMphase).Itisabsentinresting(G0)cells.Ki67antibodiesareusefulinestablishingthecellgrowingfractioninnormal,neoplasticandregenerativetissues.
Imageshowsimmunohistochemicalstainingofparaffin‐embeddedhumanglioblastomaxenografttumorsectionstainedwithKi67antibodyusingtheEtonBio’sKi67IHCKit(CatNo.3100010105).Ki67(darkbrown)displaysanuclearlocalizationpatternwhichcorrelatesitsfunctionincycleprogression(20X,counterstainedwithhematoxylin).
KitContents
Reagentsprovidedinthekit
Thematerialslistedaresufficientfor20tests.Thenumberoftestsisbasedontheuseof200μLeachofready‐to‐usereagentperslide.
PositiveControlSlides
•Onehumanrenalcarcinomaslides
BlockingBuffer
•10XNon-specificblockingbuffer
•Diluteat1:10usingdistilledwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.
EquilibriumBuffer
•EquilibriumBuffer
•Ready‐to‐usereagent
Rabbitanti-Ki67antibody
•Rabbitanti-Ki67antibody
•DiluteinAntibodyDiluentsimmediatelybeforeuse(recommenduseat1:100dilution).
AntibodyDiluent
•AntibodyDilutent
•Ready‐to‐usereagent
WashBuffer
•TrisbufferedsalinewithTween20(pH7.6)
•Diluteat1:20usingdistilledwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.
RabbitHRPPolymer
•RabbitHRPPolymer
•Ready‐to‐usereagent
DABsubstratebuffer
•10XDABsubstratebuffer
HydrogenPeroxide(H2O2)forDABsubstratebuffer
•0.3%HydrogenPeroxidesolution
DABChromogen
•Diaminobenzedinetetrahydrochloride(DAB)substratesolution(DonotexposeDABcomponentstodirectorbrightlightduringstorageandstainingprocess).
Materialsrequiredbutnotincludedinthekit
Reagents:
•Xylene
•Ethanol
•EndogenousPeroxidaseBlockingSolution(3%HydrogenPeroxide)
•Hematoxylin
•Mountingmedia
•Distilledordeionizedwater
•AntigenRetrievalBuffer(10X)0.1MCitrateBuffer(pH6.0)
LabEquipment:
•Steamerormicrowaveoven(forantigenretrieval)
•PAPpenforrestrainingreagentsonslides
•Moistchamberforslidesincubationwithstainingreagents
•Generallabequipmentforimmunohistostainingsuchasslideracks,stainingjars,coverslips,timer,Pipettes,etc.Microscopeequipmentandaccessories
StorageandstABIlity
StoreKi67IHCKitsat2‐8°C.Thekitisstableforsixmonthsat4°C.Donotuseafterexpirationdate.
Precautions
Takereasonableprecautionswhenhandlingreagents.UsedisposablegloveswhenhandlingsUSPectedcarcinogensortoxicmaterials(examples:DAB,xyleneandH2O2).Unusedsolutionshouldbedisposedaccordingtoapplicablelocal,stateandfederalregulations.
TheKi67ImmunohistostainingKithasbeendesignedforthestainingoftissuesthathavebeenfixed(usuallyinneutralbufferedformalin)andsubsequentlyembeddedinparaffinbeforesectioning.Thisprotocolisrecommendedasastartingpointandoptimizationbytheindividualend‐usermayberequired.
·Donotallowspecimenstodryduringthestainingprocedure.Specimendryingmaycauseincreasednon‐specificstainingandbackground.
·Sometissuemayneedtobaketoremoveover‐coveredparaffinpriortotheprocedure.Ifneeded,bakeat55‐60°Cfor30minutes.
Problems | PossIBLeCauses | Solutions |
Overstaining | 1.Toolongincubationtimeofprimaryantibody,ortoohightemperaturewhendoingstaining 2.ToolongincubationtimeofDABsubstrate. 3.Slidedriedduringstainingprocess | •Dependingontissuesections,theincubationtimeofprimaryantibodycanbereducedto2hours;Checktheroomtemperaturerangeisat20-250Cwhendoingstaining. •ReduceincubationtimeofDABsubstrate •Avoidsectionstodryduringstainingprocess. |
Weakornostaining | 1.Incompleteremovalofparaffin 2.Tissuesover‐fixation 3.Notefficientantigenretrieval 4.Reagentsnotusedinproperorderoromittedsteps 5.Expiredantibodyorreagents | •Deparaffinizesectionslongerorchangetofreshxylene;sometissuearraymayneedtobaketo removeover‐coveredparaffin. •Increasingtheconcentrationofprimaryantibodyto1:40;ifthisdoesnotwork,reducedurationof post‐fixation. •Adjustantigenretrievaltimebasedonthesettingforsectionfixationandretrievaldeviceused. •Reviewnotesandprocedureused. •Checkkitexpirationdatesandkitstorageconditions |
Highbackground | 1Sectionsdriedduringstainingprocess 2Slidenotrinsedthoroughly 3Antigenover‐retrieval | •Donotallowsectionstodryduringstainingprocess;usehumidcontainerduringincubation withprimaryantibody. •Usefreshsolutioninbufferjars;rinseatleastthreetimesbetweensteps. •Optimizeantigenretrievaltimeifyouusedmicrowaveorpressurecookerforretrieval. |
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一、免疫组化试剂盒一般包括：
1、特异性的一抗
2、免疫组化检测系统。
3、有的同时还具备显色系统。
二、不同的公司内容有差别，所以购买时一定了解清楚：
1、一抗是做人的还是兔、鼠的组织；
2、检测系统是ABC/SP/非生物素/其他；
3、试剂盒的内容都有什么？有没有显色系统？
4、如果没有，自己根据前面的检测系统需配哪个显色系统。（其他的辅助试剂如PBS缓冲液、抗原修复液等一般另卖或自配）
三、免疫组化抗体即指特异性一抗，是用来标记的指标，免疫组化过程的其他试剂一律另外配备。
另外：做实验时，如遇出结果困难，记得做阳性片对照，敢于怀疑检测系统或显色系统的问题。
用双氧水去除过氧化物酶时，我是自己配的3%双氧水滴到片上而非将片浸入双氧水，这样可否？
用博士德的DAB染色，配制方法为：1mlH20中加入DAB、H2O2、TBS各一滴，我配制的时候发现将DAB加入H20时很容易沉淀，要震荡才能混匀，且将配好的DAB滴到玻片上时，DAB会结为很小块的微颗粒悬浮在液体中但并不染色，是否DAB有问题？
现在打算换中衫的试剂盒，代理商说中衫的试剂盒包括了二抗和DAB，想请教一下该试剂盒的具体内容有些什么？
万分感谢！
上海好多的供应商的，具体还是看你选择了，其实试剂盒产品都差不多，什么远慕生物，古朵生物...好多的额
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