StainingprotocolsforplacentalexplantculturesWholemountstaining.Theprotocolgivenusesasingleprimaryantibodywithnuclearcounterstaining,butitcanbeextendedtodoubleantibodystaining.ItrequiresaninvertedmicroscopewithfluorescenceIlluminationandlongworkingdistanceobjectives.Wholemountstaininginmultiwellplates.Method

1. WashtheculturesgentlyandthoroughlyinPBStwice.
2. Fixin1mlMeOH.Replacethefirstsolutionimmediatelywithasecondaliquot.Incubate30min.
3. WashthreetimeswithPBS.
4. Atthisstageculturescanbetrimmedunderthedissectingmicroscopewithasmallpairofscissors.Removepartsoftheexplantthatarenotparticipatinginanchoragetothegel.Takecarenottodamagethegelsurface.Iffloatingvilliareremovedthereislesslikelihoodofdamagingthecultureduringprocessing.
5. Incubateovernight(atleast)at4°Cinblockingsolution(4%BSA/PBS).Include0.02%sodiumazideiftheplatesaretobekeptlonger.
6. 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.
7. WashinPBS,thenovernightonaplatformshakerinPBS/BSA.
8. Incubatefor30min-2hinfluorescent2ndantibodydilutedinPBS/BSA.Platesshouldbewrappedinfoil.
9. WashovernightinseveralchangesPBSusingaplatformshaker.Culturescanbeinspectedundertheinvertedmicroscopeatanystagetomonitortheremovalofbackgroundfluorescenceinthegel.Keeptheplatesinthedarkthroughout.
10. CounterstainnucleiusingegpropidiumiodideorDAPIat5mg/mlinPBS,30min.PIorDAPIaremadeupinwaterasastocksolutionof5mg/ml.
11. WashfurtherinPBS.

1. FixasdescribedinthepreviousprotocolandrehydrateinPBS.
2. Trimthegelusingascalpelretainingthecentralareawithexplant.
3. Gentlyteasetheremaininggeloffthewellsurfaceusingasmallspatula.
4. Useapastettetotransferittoa0.5mlEppendorftube.
5. Addblockingsolution(PBS/4%BSA)andproceedasinProtocol3.Incubationscanbecarriedoutonarollermixer.
6. Afterstaining,transferthegelfragmenttoamicroscopeslideandremoveexcessPBSbyblottingwithatissue.Orientitasduringculture.
7. Encasethegelfragmentinaqueoushardeningmountant(Histotec,SEROtec,UK).Donotuseacoverslip.Incubateatroomtemperatureovernightinthedark.
8. Theculturecannowbeinspecteddirectlyusingwateroroilimmersionobjectivesandeitheruprightorinvertedoptics.

1. Trimtheedgesofthegelwithascalpelifthecultureisconfinedtoonepartofthecollagendrop.GentlyteasethegelcarryingtheculturefromthefloorofthewellandtransferretoacryotubecontainingadropofOCTcompound.AfurtherdropofOCTisplacedovertheculture,thenthetubeplacedinliquidnitrogen.Itisconvenienttokeepthetissuenearthetopofthecryotube.Liquidnitrogen-cooledisopentanemayalsobeusedforfreezing.Tissuemaybekeptfrozenforseveralweeks.
2. Precoatcleanedglassmicroscopeslideswithpoly-L-lysineat100mg/ml.
3. MountthetissueonastubusingOCTasadhesive.
4. Cut7mmsectionsusingacryostat.Airdrythesectionsthenplaceinatrayorbox,wrapinfoilandstoreat-80°C.SectionsshouldbeusedassoonaspossIBLeandcertainlywithin2months.
5. Allowsectionstowarmtoroomtemperaturethenfixwithdryacetonefor10min.RehydratesectionsusingPBSthenincubateinproteinblockingsolution(Dako;10min).
6. Standardimmunostainingprotocolsmaybeusedwithperoxidaseorfluorescentconjugates(14).Notethatalkalinephosphataseconjugatesshouldbeusedwithcautionasthisenzymeisexpressedbysometrophoblast.

• 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

1. RinsetheexplantcultureinPBS,thenfixinfreshlyprepared2.5%glutaraldehydein0.1MsodiumcacodylatebufferpH7.3for2-3hoursatroomtemperature.
2. Rinsein0.1MsodiumcacodylatebufferpH7.3with3mMCaCl2threetimesover24handstoreat4°Cuntilreadyforprocessing.Then,usingadissectingmicroscopeandrazorblade,trimthegelaroundtheexplantintoarectangularshapeabout4x8mm,ensuringthatoneshortedgeiscutatrightanglesacrosstheexplantgrowth.Thiswillbethefacethatissectionedandthemaximumareaofoutgrowthpossibleisdesirable.Transfertoaglassvialforprocessing.
3. Post-fixin1%osmiumtetroxidein0.05MsodiumcacodylatebufferpH7.3(equalparts2%aqueousOsO4solutionand0.1Mbuffer)at4°Cfor1hinaclosedglassvial.Prepareinafumecupboardandwearprotectivegloves.Donotinhalevapour.Afterfixation,decantthefixandrinsein0.1MsodiumcacodylatebufferpH7.3.
4. Dehydrateinanascendingalcoholseries:15mineachin50,70,95,100,100%ethanol.
5. Incubateinpropyleneoxide2x15min,inafumecupboard,wearingprotectivegloves.
6. Infiltrateinequalpartsepoxyresinandpropyleneoxidefor1h(Epon,Araldite,TaabEmbeddingResinorotherepoxyresinmixturescanbeused).Thismustbedoneinafumecupboard,wearinggloves.Donotinhalevapour.Followthemanufacturer"sinstructionsforthepreparationofresin.
7. Leaveovernightinclosedvialsat4°Conarotatorinamixtureofthreepartsresinand1partpropyleneoxide.
8. Nextday,givethreechangesofresinat48°Candthenembedinrectangularsiliconerubbermouldsapproximately6x11x5mmdeep.Placethegelsothatthecutsurfaceoftheexplantisupagainstthe6mmedgeofthemould,whichwillbethecuttingface.Fillthemouldstothetopwithresin.Polymeriseat60°Cfor72hours.
9. Witharazorbladeorusinganultramicrotometrimmingfacility,trimintotheblockfaceuntiltheoutgrowthisreached.Thiscanbeseenasafineblackline,andthecutvillussurfaceasaroundprofile.Thencut0.5µmthicksectionsandmountonaglassslide.Leaveonahotplate(70-80°C)todryandstainwith1%toluidinebluein1%borax.Washandexamine.
10. Suitableareascanbephotographedandthenultrathinsectionscutwithaglassordiamondknifeandmountedontocoppergrids,contrastedwithuranylacetate/leadcitrateandexaminedintheelectronmicroscope.

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