G60科创走廊
来自 : 蚂蚁淘
StainingprotocolsforplacentalexplantculturesWholemountstaining.Theprotocolgivenusesasingleprimaryantibodywithnuclearcounterstaining,butitcanbeextendedtodoubleantibodystaining.ItrequiresaninvertedmicroscopewithfluorescenceIlluminationandlongworkingdistanceobjectives.Wholemountstaininginmultiwellplates.Method- WashtheculturesgentlyandthoroughlyinPBStwice.
- Fixin1mlMeOH.Replacethefirstsolutionimmediatelywithasecondaliquot.Incubate30min.
- WashthreetimeswithPBS.
- Atthisstageculturescanbetrimmedunderthedissectingmicroscopewithasmallpairofscissors.Removepartsoftheexplantthatarenotparticipatinginanchoragetothegel.Takecarenottodamagethegelsurface.Iffloatingvilliareremovedthereislesslikelihoodofdamagingthecultureduringprocessing.
- Incubateovernight(atleast)at4°Cinblockingsolution(4%BSA/PBS).Include0.02%sodiumazideiftheplatesaretobekeptlonger.
- Incubatein1stantibodyfor30minatroomtemperature.Ideallythecultureshouldbesubmerged.Ifnecessarythevolumecanbeminimised(eg100ml)byrepeatedlypipettingthesolutionovertheexplant,preventingitfromdryingout.Antibodyshouldbedilutedin4%BSA/PBS.Agoodpositivecontrolantibodyisanti-cytokeratin7(OV-TL12/30;Dako;useat1/50).Thisidentifiestheoutgrowingcellsunequivocallyastrophoblast.Anticytokeratin8/18canbeusedformarkingcells(e.gCAM5.2,Beckton-Dickenson)butbewarethatsomeplacentalfibroblastsexpressthisMarkersoitisnotspecificfortrophoblast.
- WashinPBS,thenovernightonaplatformshakerinPBS/BSA.
- Incubatefor30min-2hinfluorescent2ndantibodydilutedinPBS/BSA.Platesshouldbewrappedinfoil.
- WashovernightinseveralchangesPBSusingaplatformshaker.Culturescanbeinspectedundertheinvertedmicroscopeatanystagetomonitortheremovalofbackgroundfluorescenceinthegel.Keeptheplatesinthedarkthroughout.
- CounterstainnucleiusingegpropidiumiodideorDAPIat5mg/mlinPBS,30min.PIorDAPIaremadeupinwaterasastocksolutionof5mg/ml.
- WashfurtherinPBS.
WholemountstainingintubesTheprotocolcanbemodifiedformoreeconomicaluseofantibodybydetachingtheculturesandstaininginEppendorftubes.Method- FixasdescribedinthepreviousprotocolandrehydrateinPBS.
- Trimthegelusingascalpelretainingthecentralareawithexplant.
- Gentlyteasetheremaininggeloffthewellsurfaceusingasmallspatula.
- Useapastettetotransferittoa0.5mlEppendorftube.
- Addblockingsolution(PBS/4%BSA)andproceedasinProtocol3.Incubationscanbecarriedoutonarollermixer.
- Afterstaining,transferthegelfragmenttoamicroscopeslideandremoveexcessPBSbyblottingwithatissue.Orientitasduringculture.
- Encasethegelfragmentinaqueoushardeningmountant(Histotec,SEROtec,UK).Donotuseacoverslip.Incubateatroomtemperatureovernightinthedark.
- Theculturecannowbeinspecteddirectlyusingwateroroilimmersionobjectivesandeitheruprightorinvertedoptics.
Cryosectioningexplantcultures.Forimmunostainingwithalargerpanelofantibodiesonthesameculture,cryosectionsarerequired.- Trimtheedgesofthegelwithascalpelifthecultureisconfinedtoonepartofthecollagendrop.GentlyteasethegelcarryingtheculturefromthefloorofthewellandtransferretoacryotubecontainingadropofOCTcompound.AfurtherdropofOCTisplacedovertheculture,thenthetubeplacedinliquidnitrogen.Itisconvenienttokeepthetissuenearthetopofthecryotube.Liquidnitrogen-cooledisopentanemayalsobeusedforfreezing.Tissuemaybekeptfrozenforseveralweeks.
- Precoatcleanedglassmicroscopeslideswithpoly-L-lysineat100mg/ml.
- MountthetissueonastubusingOCTasadhesive.
- Cut7mmsectionsusingacryostat.Airdrythesectionsthenplaceinatrayorbox,wrapinfoilandstoreat-80°C.SectionsshouldbeusedassoonaspossIBLeandcertainlywithin2months.
- Allowsectionstowarmtoroomtemperaturethenfixwithdryacetonefor10min.RehydratesectionsusingPBSthenincubateinproteinblockingsolution(Dako;10min).
- Standardimmunostainingprotocolsmaybeusedwithperoxidaseorfluorescentconjugates(14).Notethatalkalinephosphataseconjugatesshouldbeusedwithcautionasthisenzymeisexpressedbysometrophoblast.
Resinembeddingofexplantcutures.Examinationofsemithinresinsectionscanshowthepatternofcellgrowthacrossandwithinthegel.Electronmicroscopyenablescell-cellandcell-matrixinteractionstobevisualised.Equipmentandreagents- Dissectingmicroscope
- Glassvialswithplasticsnap-ontops.
- Oven/incubator@48°and60°C
- Siliconerubbermoulds(AgarScientificLtd,TaabLaboratoriesEquipmentLtd)
- 25%glutaraldehyde(EMgrade)(AgarScientificLtd,TaabLaboratoriesEquipmentLtd)
- 2%aqueousosmiumtetroxide(AgarScientificLtd,TaabLaboratoriesEquipmentLtd)
- Propyleneoxide
- Epoxyresin(AgarScientificLtd,TaabLaboratoriesEquipmentLtd)
- 0.1MsodiumcacodylatebufferpH7.3+/-3mMcalciumchloride
Method- RinsetheexplantcultureinPBS,thenfixinfreshlyprepared2.5%glutaraldehydein0.1MsodiumcacodylatebufferpH7.3for2-3hoursatroomtemperature.
- Rinsein0.1MsodiumcacodylatebufferpH7.3with3mMCaCl2threetimesover24handstoreat4°Cuntilreadyforprocessing.Then,usingadissectingmicroscopeandrazorblade,trimthegelaroundtheexplantintoarectangularshapeabout4x8mm,ensuringthatoneshortedgeiscutatrightanglesacrosstheexplantgrowth.Thiswillbethefacethatissectionedandthemaximumareaofoutgrowthpossibleisdesirable.Transfertoaglassvialforprocessing.
- Post-fixin1%osmiumtetroxidein0.05MsodiumcacodylatebufferpH7.3(equalparts2%aqueousOsO4solutionand0.1Mbuffer)at4°Cfor1hinaclosedglassvial.Prepareinafumecupboardandwearprotectivegloves.Donotinhalevapour.Afterfixation,decantthefixandrinsein0.1MsodiumcacodylatebufferpH7.3.
- Dehydrateinanascendingalcoholseries:15mineachin50,70,95,100,100%ethanol.
- Incubateinpropyleneoxide2x15min,inafumecupboard,wearingprotectivegloves.
- Infiltrateinequalpartsepoxyresinandpropyleneoxidefor1h(Epon,Araldite,TaabEmbeddingResinorotherepoxyresinmixturescanbeused).Thismustbedoneinafumecupboard,wearinggloves.Donotinhalevapour.Followthemanufacturer"sinstructionsforthepreparationofresin.
- Leaveovernightinclosedvialsat4°Conarotatorinamixtureofthreepartsresinand1partpropyleneoxide.
- Nextday,givethreechangesofresinat48°Candthenembedinrectangularsiliconerubbermouldsapproximately6x11x5mmdeep.Placethegelsothatthecutsurfaceoftheexplantisupagainstthe6mmedgeofthemould,whichwillbethecuttingface.Fillthemouldstothetopwithresin.Polymeriseat60°Cfor72hours.
- Witharazorbladeorusinganultramicrotometrimmingfacility,trimintotheblockfaceuntiltheoutgrowthisreached.Thiscanbeseenasafineblackline,andthecutvillussurfaceasaroundprofile.Thencut0.5µmthicksectionsandmountonaglassslide.Leaveonahotplate(70-80°C)todryandstainwith1%toluidinebluein1%borax.Washandexamine.
- Suitableareascanbephotographedandthenultrathinsectionscutwithaglassordiamondknifeandmountedontocoppergrids,contrastedwithuranylacetate/leadcitrateandexaminedintheelectronmicroscope.
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