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).
Reagentsprovidedinthekit
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.
BlockingofNon-specificbinding
Note:10Xblockingbuffermayformprecipitatesat4°C.Completelydissolvetheprecipitatesbeforemakingworkingsolution
PrimaryAntibody
Secondary/HRPConjugates
DABChromogen
13.TapoffexcessPBS.ApplyenoughDABSubstrateSolutiontocoverspecimenimmediatly.Checkdarkbrowncolordevelopmentundermicroscopeandincubateuntildesiredstainintensitydevelops.
DistilledWater--860μL
10XDABsubstratebuffer--100μL
0.3%HydrogenPeroxidesolution--15μL
DABChromogen--25μL
Counterstaining
Mounting
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. |
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一、免疫组化试剂盒一般包括：
1、特异性的一抗
2、免疫组化检测系统。
3、有的同时还具备显色系统。
二、不同的公司内容有差别，所以购买时一定了解清楚：
1、一抗是做人的还是兔、鼠的组织；
2、检测系统是ABC/SP/非生物素/其他；
3、试剂盒的内容都有什么？有没有显色系统？
4、如果没有，自己根据前面的检测系统需配哪个显色系统。（其他的辅助试剂如PBS缓冲液、抗原修复液等一般另卖或自配）
三、免疫组化抗体即指特异性一抗，是用来标记的指标，免疫组化过程的其他试剂一律另外配备。
另外：做实验时，如遇出结果困难，记得做阳性片对照，敢于怀疑检测系统或显色系统的问题。
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！
上海好多的供应商的，具体还是看你选择了，其实试剂盒产品都差不多，什么远慕生物，古朵生物...好多的额
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