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蚂蚁淘在线 / 品牌中心 / Everest Biotech / Everest Biotech/Ki67 Immunohistochemistry Kit 10 slides/3100010105/10 slides

Everest Biotech/Ki67 Immunohistochemistry Kit 10 slides/3100010105/10 slides

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￥3473.60

货号：3100010105

浏览量：127

品牌：Everest Biotech

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商品描述

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.

Note:

·Donotallowspecimenstodryduringthestainingprocedure.Specimendryingmaycauseincreasednon‐specificstainingandbackground.

·Sometissuemayneedtobaketoremoveover‐coveredparaffinpriortotheprocedure.Ifneeded,bakeat55‐60°Cfor30minutes.

I.Deparaffinizationandrehydration

Priortostaining,tissuesectionsmustbedeparaffinizedandrehydrated.Incompleteremovalofparaffincancausepoorstainingofthesection.Usepositivecontrolslideprovidedinthekitforqualitycontrolandtrouble-shootingpurpose.

1.Immerseslidesinxyleneandincubatefor5minutes.Repeattwicewithfreshxyleneforanother5minuteseach.

2.Immerseslidesin100%ethanolfor5minutes,andfollowwithimmersionin95%,75%and50%ethanolfor3minuteseach.

3.Rinseslideswithdistilledwaterfor5minutes;keepinwateruntilreadytoperformantigenretrieval.

II.Heatinducedantigenretrieval(HIAR)

Mostformalin‐fixedtissuerequiresanantigenretrievalstepbeforeimmunohistochemicalstainingcanproceed.Heatinducedantigenretrievalcanbeperformedusingasteamer,pressurecooker,oramicrowaveoven.Theretrievaltimewritteninthisprotocolisbasedonusingasteamer.Theheatingtimemayneedtobeadjustedifyouuseadifferentdeviceandmethod.

1.Fillplasticcoplinjar/containerwithAntigenRetrievalBuffer(0.01MCitrateBuffer,pH6.0,notincludedinthekits).

PrepareStockSolution:

A.0.1MSodiumCitrate;

B.0.1MCitricAcid

Tomake250mL1Xantigenretrievalbuffer,mix20.5mLstocksolutionAwith4.5mLstocksolutionBandadddistilledwaterto250mL

2.Placethecoplinjar/containerinsteamerwithlid.

3.Turnonsteamerandpreheatto90‐100°C.Carefullyputslidesintothecoplinjar/containerandsteamfor40min(95‐100°C).

4.Turnoffthesteamer,removethecoplinjar,placeatroomtemperatureandallowslidestocoolfor20min.Keepthejarcoveredallthetime.

5.Rinseslidebyincubationofslideindistilledwaterfor3minutes.Repeatthissteptwiceandbeginstainingprocedure.

III.Stainingprocedure

BlockingofEndogenousPeroxidase

Note:PeroxidaseBlockingisoptional.Ifnonon-specificstainingisobserved,skipthesestepsandgotostep3.

1.Tapoffexcesswater.DrawacirclearoundthespecimenontheslidewithPAPpen(notincludedinthekit.Alternatively,foldedKimwipescouldbeusedtobrieflyblotthewateraroundthespecimen.Repeatthisblotstepeachtimebeforeaddreagentonslide).Apply200μlormoreofPeroxidaseBlockingSolution(notincludedinthekit)sufficienttocoverspecimen,andincubatefor5minutes.

2.Rinseslidebyincubationofslideindistilledwaterfor3minutes.Repeatthissteptwice.

3.RinseslidebyincubationslideinPBSfor3minutes.

BlockingofNon-specificbinding

4.TapoffexcessPBS.(IfthePeroxidaseBlockingstepisskipped,drawacirclearoundthespecimenontheslidewithPAPpenorusingtheedgeoffoldedKimwipestoquicklyblotthewateraroundthespecimen).Apply200μl1XBlockingBufferimmediatelytocoverspecimenandincubateinamoistchamberfornomorethan10minutes

Note:10Xblockingbuffermayformprecipitatesat4°C.Completelydissolvetheprecipitatesbeforemakingworkingsolution

5.RinseslidebyincubationslideinPBSfor3minutes.

PrimaryAntibody

6.TapoffexcessPBS.Apply200μlEquilibriumBufferimmediatelytocoverspecimenandincubateinamoistchamberfor30minutes

7.TapoffexcessEquilibriumBuffer.Apply200μlanti‐Ki67antibody(recommend1:100dilutioninAntibodyDiluent)tocoverspecimenimmediatelyandincubateinamoistchamberovernightat4°C.

8.Rinseslidebyincubationin0.5-2mLWashBufferfor3minutes.Repeatthissteptwicewithfreshbuffer.

9.RinseslidebyincubationofslideinPBSfor3minutes.

Secondary/HRPConjugates

10.TapoffexcessPBS.Apply200μlRabbitHRPPolymerimmediatelytocoverspecimenandincubateinamoistchamberfor60minutes.

11.Rinseslidebyincubationin0.5-2mLWashBufferfor3minutes.Repeatthissteptwicewithfreshbuffer.

12.RinseslidebyincubationinwithPBSfor3minutes.

DABChromogen

13.TapoffexcessPBS.ApplyenoughDABSubstrateSolutiontocoverspecimenimmediatly.Checkdarkbrowncolordevelopmentundermicroscopeandincubateuntildesiredstainintensitydevelops.

Tomake1mLDABSubstrateSolution,mixthefollowingreagents:

DistilledWater860μL

10XDABsubstratebuffer100μL

0.3%HydrogenPeroxidesolution15μL

DABChromogen25μL

14.Rinseslideintapwaterfor3minutes.

Counterstaining

15.Ifdesired,completecounterstain(Seeinstructionforhematoxylincounterstaining).Rinseintapwatertoclear.

Mounting

16.Immerseslidesin70%,80%,95%Ethanolfor2minuteseach,and100%Ethanolfor10minutestwicefollowedbyXylenefor5minutestwice.

17.Dryandmountslides.

IV.InstructionforHematoxylincounterstaining

1.Immerseslidesinhematoxylinsolution.Incubatefor30secondsto5minutes,dependingonthestrengthofhematoxylinused.

2.RinsetoclearwithtapwaterandcontinuedehydrationfromStep16.

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.

Everest Biotech自2000年以来，我们一直是抗肽和抗原亲和纯化山羊多克隆抗体的专家。我们的抗体具有100％的满意保证-它们可在您的实验室或您的退款中发挥作用。 LifeSpan BioSciences，Inc.收购了Everest Biotech。在此处查找使用Everest抗体的最新出版物盖玻片用于悬浮细胞免疫荧光

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