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蚂蚁淘在线 / 品牌中心 / Everest Biotech / Everest Biotech/Cyclin D1 Immunohistochemistry Kit 20 slides/3100050205/20 slides

Everest Biotech/Cyclin D1 Immunohistochemistry Kit 20 slides/3100050205/20 slides

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￥5189.60

货号：3100050205

浏览量：127

品牌：Everest Biotech

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Duringthecellcycleofmostsomaticcells,DNAsynthesis(S-phase)andmitosis(M-phase)areseparatedbytwogapphases(G1andG2)ofvaryingduration.Regulationofcellcycleprogressionineukaryoticcellsdependsontheexpressionofcyclinproteins.Membersofthecyclinfamilyofproteinscombinewithacyclindependentkinases(CDKs)subunittoformtheactivekinase,whichinitiatesG2toMandG1toStransition.ThelatterarecontrolledbycyclinstermedG1cyclins,whichcommitthecelltoDNAreplication.AtleastfivecandidateG1-phasecyclins,termedcyclinsC,D1,D2,D3,andEhavebeenidentifiedinmammaliancells.D-typecyclinsareinducedduringtheG1phaseofthemammaliancellcycleinresponsetoavarietyofmitogenicgrowthfactors.Onceinduced,theD-typecyclinsaccumulateincomplexeswithCDKs,whosekinaseactivityisthoughttobenecessaryfordrivingcellsintoSphase.ThemajorcatalyticpartnersoftheD-typecyclinsareCDK4andCDK6,butatleastsomeD-typecyclinsalsointeractwithotherCDKs,includingCDK2andCDK5.CyclinD1-andD2-associatedCDK4and/orCDK6kinaseactivitieshavebeendetectedinmid-G1,priortotheactivationofanyotherknownCDK,andtheyculminateinlateG1phase.AmplificationofthecyclinD1genehasbeenobservedinasignificantpercentageofcancers,includingbreast,squamous,andesophagealcarcinomas.

Imageshowsimmunohistochemicalstainingofparaffin‐embeddedhumanbreastadenocarcinomaxenografttumorsectionstainedwithCyclinD1antibodyusingtheEtonBio"sCyclinD1IHCKit.CyclinD1(darkbrown)displaysanuclearlocalizationpatternwhichcorrelatesitsfunctioninregulatingcycleprogression(20X,counterstainedwithhematoxylin).

KitContents

Reagentsprovidedinthekit

Thenumberoftestsisbasedontheuseof200μLeachofready-to-usereagentperslide.

PositiveControlSlide

•positivecontrolslide

EquilibriumBuffer

•EquilibriumBuffer

•Ready-to-usereagent

BlockingBuffer

•10XNon-specificblockingbuffer

•Diluteat1:10usingdistilledwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.

RabbitcyclinD1antibody

•Rabbitanti-cyclinD1antibody

•DiluteinAntibodyDiluentsimmediatelybeforeuse(recommenduseat1:50dilution).

AntibodyDiluent

•AntibodyDilutent

•Ready-to-usereagent

WashBuffer(20x)

•TrisbufferedsalinewithTween20(pH7.6)

•Diluteat1:20usingdistilledordeionizedwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.

RabbitHRPPolymer

•RabbitHRPPolymer

•Ready-to-usereagent

DABsubstratebuffer

•10XDABsubstratebuffer

•Beforeuse,add100μL10XDABsubstratebufferto1mLofdistilledordeionizedwatertomake1XDABsubstratebuffer.

HydrogenPeroxide(H₂O₂)forDABsubstratebuffer

•0.3%HydrogenPeroxidesolution

•Beforeuse,add15μLH₂O₂to1mLof1XDABsubstratebuffer.

DABChromogen

•Diaminobenzedinetetrahydrochloride(DAB)substratesolution

•DABSubstrateSolution

Materialsrequiredbutnotincludedinthekit

Reagents:

•Xylene

•Ethanol

•EndogenousPeroxidaseBlockingSolution(3%HydrogenPeroxide)

•Hematoxylin

•Mountingmedia

•Distilledordeionizedwater

•AntigenRetrievalBuffer(10X)0.1MCitrateBuffer(pH6.0)Diluteat1:10using

LabEquipment:

•Steamerormicrowaveoven(forantigenretrieval)

•Generallabequipmentforimmuno-histostainingsuchasslideracks,stainingjars,coverslips,timer,Pipettes,etc.Microscopeequipmentandaccessories

StorageandstABIlity

StoreIHCKitsat2‐8°C.Thekitisstableforsixmonthsat4°C.

Donotuseafterexpirationdate.

Precautions

Takereasonableprecautionswhenhandlingreagents.UsedisposablegloveswhenhandlingsUSPectedcarcinogensortoxicmaterials(examples:DAB,xyleneandH₂O₂).Unusedsolutionshouldbedisposedaccordingtoapplicablelocal,stateandfederalregulations.

TheCyclinD1ImmunohistostainingKithasbeendesignedforthestainingoftissuesthathavebeenfixed(usuallyinneutralbufferedformalin)andsubsequentlyembeddedinparaffinbeforesectioning.Thisprotocolisrecommendedasastartingpoint.Wheneverusinganewantibodyorimmunohistochemistrykit,optimizationbytheindividualend-usermayberequired.

Note:

•Donotallowspecimenstodryduringthestainingprocedure.Specimendryingmaycauseincreasednon-specificstainingandbackground.

•Sometissuemayneedtobaketoremoveover-coveredparaffinpriortotheprocedure.Ifneeded,bakeat55-60°Cfor30minutes.

I.Deparaffinizationandrehydration

Priortostaining,tissuesectionsmustbedeparaffinizedandrehydrated.Incompleteremovalofparaffincancausepoorstainingofthesection.

Immerseslidesinxyleneandincubatefor5minutes.Repeattwicewithfreshxyleneforanother5minuteseach.
Immerseslidesin100%ethanolfor5minutes,andfollowwithimmersionin95%,75%and50%ethanolfor3minuteseach.
Rinseslideswithdistilledwaterfor5minutes;keepinwateruntilreadytoperformantigenretrieval.

II.Heatinducedantigenretrieval(HIAR)

Mostformalin-fixedtissuerequiresanantigenretrievalstepbeforeimmunohistochemicalstainingcanproceed.Heatinducedantigenretrievalcanbeperformedusingasteamer,pressurecooker,oramicrowave.Theretrievaltimewritteninthisprotocolisbasedonusingaretrievalsteamer.Theheatingtimemayneedtobeadjustedifyouuseadifferentdeviceandmethod.

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

PrepareStockSolution:

0.1MSodiumCitrate--20.5mL

0.1MCitricAcid--4.5mL

Adddistilledwaterto250mL

2.Placethecoplinjar/containerinsteamerwithlid.
3.Turnonsteamerandpreheatto90‐100°C.Carefullyputslidesintothecoplinjar/containerandsteamfor40min(95‐100°C).
4.Turnoffthesteamer,removethecoplinjar,placeatroomtemperatureandallowslidestocoolfor20min.Keepthejarcoveredallthetime.
5.Rinseslidebyincubationofslideindistilledwaterfor3minutes.Repeatthissteptwiceandbeginstainingprocedure.

III.Stainingprocedure

PeroxidaseBlockingisoptional.Ifnonon-specificstainingisobserved,skipthesestepsandgotostep3.

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.Apply200μldilutedanti‐CyclinD1antibody(recommend1:50dilutioninAntibodyDiluent)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:

DistilledWater--860μL

10XDABsubstratebuffer--100μL

0.3%HydrogenPeroxidesolution--15μL

DABChromogen--25μL

14.Rinseslideintapwaterfor3minutes.

Counterstaining

15.Ifdesired,completecounterstain(Seeinstructionforhematoxylincounterstaining).Rinseintapwatertoclear.

Mounting

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

17.Dryandmountslides.

IV.InstructionforHematoxylincounterstaining

Immerseslidesinhematoxylinsolution.Incubatefor30secondsto5minutes,dependingonthestrengthofhematoxylinused.
RinsetoclearwithtapwaterandcontinuebydehydrationfromStep14.

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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