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‐embeddedhumanbreastcancerxenografttumorsectionstainedwithAKTThr308antibodyusingtheEtonBio’sAKTThr308IHCKit.AKTThr308(darkbrown)displaysatumorcellstainingpattern(20X,counterstainedwithhematoxylin).
KitContents
Reagentsprovidedinthekit
Thematerialslistedaresufficientfor20tests.Thenumberoftestsisbasedontheuseof200μLeachofready‐to‐usereagentperslide.
PositiveControlSlides
•Onehumanrenalcarcinomaslides
BlockingBuffer
•10XNon-specificblockingbuffer
•Diluteat1:10usingdistilledwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.
EquilibriumBuffer
•EquilibriumBuffer
•Ready‐to‐usereagent
Rabbitanti-pAKT308antibody
•Rabbitanti-pAKT308antibody
•DiluteinAntibodyDiluentsimmediatelybeforeuse(recommenduseat1:200dilution).
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
StorepAKT308IHCKitsat2‐8°C.Thekitisstableforsixmonthsat4°C.Donotuseafterexpirationdate.
Precautions
Takereasonableprecautionswhenhandlingreagents.UsedisposablegloveswhenhandlingsUSPectedcarcinogensortoxicmaterials(examples:DAB,xyleneandH2O2).Unusedsolutionshouldbedisposedaccordingtoapplicablelocal,stateandfederalregulations.
ThePAKT308ImmunohistostainingKithasbeendesignedforthestainingoftissuesthathavebeenfixed(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‐pAKT308antibody(recommend1:200dilutioninAntibodyDiluent)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. |
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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！
蛋白的类型为PDGF。其有一种二聚体为PDGF-BB。
请教第一点：我看了Elisa的说明书，现在检测PDGF有两种试剂盒，一种是检测PDGF的，要求的试剂样本为组织匀浆；一种是检测PDGF-BB的，要求的待测样本为血液或血浆。
我现在的实验目的是想更精确，检验组织中的PDGF-BB，但是手中的是人体韧带组织样本，非PDGF-BB试剂盒所要求的组织样本为血浆或血清，我不知道是否可以用PDGF-BB的ELISA试剂盒的来做，所以特地请教：）
请教第二点：市面上有RB公司的96T，2500元；另外有Uscnlife的Elisa试剂盒，96T的3680元，都说是原装进口的，我该如何选择呢？为何差价这么多？谢谢指点！
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