Theactivationofsignaltransductionpathwaysbygrowthfactors,hormonesandneurotransmittersismediatedthroughtwocloselyrelatedMAPkinases(p44andp42),designatedERK1(extracellular-signalrelatedkinase1)andERK2,respectively.Uponstimulation,asequentialthree-partproteinkinasecascadeisinitiated,consistingofaMAPkinasekinasekinase(MAPKKKorMAP3K),aMAPkinasekinase(MAPKKorMAP2K),andaMAPkinase(MAPK).Multiplep44/42MAP3Kshavebeenidentified,includingmembersoftheRaffamilyaswellasMosandTpl2/Cot.MEK1andMEK2aretheprimaryMAPKKsinthispathway.MEK1andMEK2activatep44andp42throughphosphorylationofactivationloopresiduesThr202/Tyr204andThr185/Tyr187,respectively.Severaldownstreamtargetsofp44/42havebeenidentified,includingp90RSKandthetranscriptionfactorElk-1.p44/42arenegativelyregulatedbyafamilyofdual-specificity(Thr/Tyr)MAPKphosphatases,knownasDUSPsorMKPs.
Imageshowsimmunohistochemicalstainingofparaffin‐embeddedhumanovariancancerxenografttumorsectionstainedwithpERKantibodyusingtheEtonBio"spERKIHCKit(CatNo.3100130105).pERK(darkbrown)displaysatumorcellstainingpattern(20X,counterstainedwithhematoxylin).
Reagentsprovidedinthekit
Thematerialslistedaresufficientfor20tests.Thenumberoftestsisbasedontheuseof200μLeachofready‐to‐usereagentperslide.
PositiveControlSlides
•Onehumanrenalcarcinomaslides
BlockingBuffer
•10XNon-specificblockingbuffer
•Diluteat1:10usingdistilledwaterpriortostaining;unusedworkingsolutionmaybestoredat4°Cfor3month.
EquilibriumBuffer
•EquilibriumBuffer
•Ready‐to‐usereagent
Rabbitanti-pERKantibody
•Rabbitanti-pERKantibody
•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
StorepERKIHCKitsat2‐8°C.Thekitisstableforsixmonthsat4°C.Donotuseafterexpirationdate.
Precautions
Takereasonableprecautionswhenhandlingreagents.Usedisposablegloveswhenhandlingsuspectedcarcinogensortoxicmaterials(examples:DAB,xyleneandH2O2).Unusedsolutionshouldbedisposedaccordingtoapplicablelocal,stateandfederalregulations.
Protocol
ThepERKImmunohistostainingKithasbeendesignedforthestainingoftissuesthathavebeenfixed(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‐pERKantibody(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 | Dependingontissuesections,theincubationtimeofprimaryantibodycanbereducedto30minutes;Checktheroomtemperaturerangeisat20‐250Cwhendoingstaining. |
Weakornostaining | 1.Incompleteremovalofparaffin | Deparaffinizesectionslongerorchangetofreshxylene;sometissuearraymayneedtobaketo |
Highbackground | 1Sectionsdriedduringstainingprocess | Donotallowsectionstodryduringstainingprocess;usehumidcontainerduringincubation |
