Everest Biotech/pAKT Ser473 Immunohistochemistry Kit 10 slides/3100100105/10 slides,Everest Biotech,,Everest Biotech,everestbiotech,+,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：Everest Biotech/pAKT Ser473 Immunohistochemistry Kit 10 slides/3100100105/10 slides

产品编号：3100100105

市场价：¥3473.60

场地：美国(厂家直采)

产品分类：试剂盒>免疫组化>免疫组化试剂盒>

联系QQ：1570468124

电话号码：4000-520-616

邮箱： info@ebiomall.com

美元价：$334.00

品牌： Everest Biotech

公司：Everest Biotech

公司分类：

商品介绍

AKT,alsoknownasproteinkinaseB(PKB),isa57kDaserine/threonineproteinkinase.TherearethreemammalianisoformsofAkt:AKT1(PKBalpha),AKT2(PKBbeta)andAKT3(PKBgamma).Eachisoformhasapleckstrinhomology(PH)domain,akinasedomainandacarboxyterminalregulatorydomain.AKTisakeyregulatorofmanysignaltransductionpathwaysandmediatesmanyofthedownstreameventsofphosphatidylinositol3kinase.AktisactivatedbyphospholipidbindingandactivationloopphosphorylationatThr308byPDK1andbyphosphorylationwithinthecarboxyterminusatSer473bymammaliantargetofrapamycin(mTOR)inarapamycin-insensitivecomplexwithrictorandSin1.Aktpromotescellsurvivalbyinhibitingapoptosisthroughphosphorylationandinactivationofseveraltargets,includingBad,forkheadtranscriptionfactors,c-Rafandcaspase-9.AnotheressentialAktfunctionistheregulationofglycogensynthesisthroughphosphorylationandinactivationofGSK-3αandβ.AktisinvolvedincellcycleregulationbypreventingGSK-3βmediatedphosphorylationanddegradationofcyclinD1andbynegativelyregulatingthecyclindependentkinaseinhibitorsp27Kipandp21Waf1/CIP1.

Imageshowsimmunohistochemicalstainingofparaffin‐embeddedhumanbreastcancerxenografttumorsectionstainedwithAKTSer473antibodyusingtheEtonBio’sAKTSer473IHCKit(CatNo.3100100105).AKTSer473(darkbrown)displaysatumorcellstainingpattern(20X,counterstainedwithhematoxylin).

KitContents

Reagentsprovidedinthekit

Thematerialslistedaresufficientfor20tests.Thenumberoftestsisbasedontheuseof200μLeachofready‐to‐usereagentperslide.

PositiveControlSlides

•Onehumanrenalcarcinomaslides

BlockingBuffer

•400μl10XNon-specificblockingbuffer

•Diluteat1:10usingdistilledwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.

EquilibriumBuffer

•EquilibriumBuffer

•Ready‐to‐usereagent

Rabbitanti-pAKT473antibody

•Rabbitanti-pAKT473antibody

•DiluteinAntibodyDiluentsimmediatelybeforeuse(recommenduseat1:50dilution).

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

StorePAKT473IHCKitsat2‐8°C.Thekitisstableforsixmonthsat4°C.Donotuseafterexpirationdate.

Precautions

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

ThePAKT473ImmunohistostainingKithasbeendesignedforthestainingoftissuesthathavebeenfixed(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.The

retrievaltimewritteninthisprotocolisbasedonusingasteamer.Theheatingtimemayneedtobeadjustedifyouuseadifferentdeviceandmethod.

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

PrepareStockSolution:

0.1MSodiumCitrate20.5mL

0.1MCitricAcid4.5mL

Adddistilledwaterto250mL

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.Apply200μldilutedanti‐pAKT473antibody(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:

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,theincubationtimeofprimaryantibodycanbereducedto30minutes;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.

品牌介绍

Everest Biotech公司成立于2000年，由英国牛津大学一批生物化学家创建。公司科学家团队具有丰富的抗体纯化及蛋白组学专业知识，雄厚的专业背景赋予了公司能为全球科研人员提供高质量、创新性抗体产品的能力。目前，Everest Biotech可提供3000余种抗体，涉及肿瘤、自身免疫、过敏、心血管疾病、传染病、代谢、神经科学等领域。所有抗体均由Everest Biotech自主研发生产、全部经过肽亲和纯化以保证最佳的特异性和纯度、经过免疫原肽的ELISA和WB实验验证; 由PhD科学家团队提供全方位技术支持服务。Everest Biotech为众多国际知名抗体公司提供OEM服务。