1)FemaleTiel-PRTgmiceandwildtypecontrollitter-matesaged6-9wkswereovariectimizedandallowedtorecoverfor7-10days.Makingestrogenandprogesteronestocksolutions:Forestrogen(17beta-estrADIolfromSigma),weighout10mgandaddto1ml200-proofEtOH.ThiswillyieldalOmg/mlsolution.Mixbyvortexing.Performa1:100dilutionbyadding5μlofthelOmg/mlsolutionto495μlEtOH.Thiswillleaveyouwiththe100μg/mlstocksolutionwhichyoucankeepat-20Cforprobablyabout2weeks.Fortheprogesteronestock:Weighout5-10mgprogesterone(Sigma)andaddtothecorrespondingamountofEtOHtoyielda1mg/mlsolution.(Forexample:6.7mgprogesteronewouldbeaddedto6.7mlEtOH).Mixbyvortexingandstoreat-20C-stableabout2weeks.2)Themiceweretreatedwiththefollowinghormoneregime:Injectmicesubcutaneously(ontheirunderside)for3days(onceadayaround10-11AM)with100ng17β-estradiol(Sigma)dissolvedin0.1mlsesameoil.1-Directions:Takea1.5mltubeandplace10μlofthelOOug/mlstocksolutionintothetube.Add990μlsesameoiltothesametubeandvorteximmediatelyandthoroughly(theEtOHwillhaveatendencytomovetothetopoftheoil).Injecting100μlwilldeliverlongestrogen.Inreality,injectabout120μltoaccountforsomebackflowleakage.Animalsreceivingvehiclegetinjectedwith100μlsesameoilalone.Makesuretogos.c.andnoti.p.Theskinshouldballoonoutifyouinjectthe100μls.c.Wettingthehairwithasmallamountof70%EtOHwillhelptheinjectionbecausetheskincanbemoreeasilyvisualized.3)OnDays4and5themicereceivednotreatment.4)Ondays6-8,micereceivedlμgprogesterone(Sigma)and6.7ng17-β-estradioiin0.1mlsesameoil(onceadayaround10-11AM).-Directions:Takea1.5mltubeandmix33.5μlofthe100μg/mlestrogenstocksolutionwith466.5μlEtOH(finalvolume500μl)tomakea1:14.93dilutionandthereforea6.7μg/mlstocksolution.Storesolutionat-20Cforatmax2weeks.Add10μleachofthe6.7μg/mlestrogenstockand1mg/mlprogesteronestocksolutionstoa1.5mltubeandthenadd980μlsesameoil.Vorteximmediatelyandthroughly.Injecting100μlwilldeliver6.7ngestrogenand1μgprogesterone.Inreality,injectabout110μ1toaccountforsomebackflowleakage.5)5-7hoursafterreceivingthelasthormonetreatmentonday8,themicewereinjectedbytailveinwith1ml/kg3%EvansBluedissolvedin0.9%salineforinjection.-Directions:removeevansbluestockbottlefrom4Candplaceonmagneticstirrerfor1-2hours.Remove3-4mlandplaceinplastictissuespecimencontainer.Sonicateonsetting5with5-10pulsesforabout1min.Keeptubeoniceandmakesureyoucoverthetopofthecontainer(withtheprobestickingoutofit)withParafilmtokeeptheE.Bfromsprayingout.FiltertheEBaftersonicationwitha0.22μmsyringefiltertoremoveanyparticlesleftinthesolution.Weighthemice(probablywillbearound20-25g)andmeasureouttheamountofEB(inμl)thatmatchestheirweight.Thenadd0.9%salineforinjectiontoafinalvolumeof100μl(easiertoinject100μlthen20-25μl).Forexamplefora25gmouse,add25μlEBto75μlsalineina1,5mltube.Pullsolutionupin0.5ccinsulinsyringe.Injectintotailveinusingplasticrestrainerandwarmingthetailin45Cwater.Notethetimeofinjection-perfusionwillbe20minsfollowinginjection.6)15minafterinjection,themicewereanesthetizedwithisofluraneusinga50mlconicalnoseconedevice.2.Around19minafterinjection,thechestcavitywasopenedtoexposetheheart,therightatriaremoved,theleftventriclecut,andperfusedat120mmHgthroughtheascendingaortawith1%paraformaldehydein0.05Mcitratebuffer,pH3.5for2min.Thetimebetweenthechestcutandthestartofperfusionwas8-15secandoptimallyoccurredatthe20mintimepoint3.-TomakethePFA:dissolvelOgPFAinabout200-300mldH20at50-60C.Onceinsolutionadd14.7gsodiumcitrate.Bringthetotalvolumeuptoabout800-900mlwithdH20.pHthesolutionto3.5(youwillneedtoaddalotofacid).TopthePFAoffwithdH20tothefinalvolumeof1L.Filtersolutionwith#50Whatmanpaper.7)Afterperfusion,thefirst4cmofintestinelocatedimmediatelydistaltothestomachwasresectedandthelumenflushedwithfixative.Theuterinehornswerealsodissectedandeachtissuewasblottedbetween2piecesofWhatmanfilterpaperfor10sec(placeafullPipettebluetipcontainerontopofpaperandcountto10)andweighed(wetweight).Thetissueswereplacedin1mloffomiamide(inlabeledglass7mlsolvent-savertypetubes)o/nat56°CtoextracttheEvansBlue.Thefollowingday,thetissueswereremovedfromtheformamideandtheA620ofthesamplesandstandardcurvewasmeasuredonaspectophotometerandtheresultswereexpressedasngEvansBlue/tissueandngEvansBlue/mgtissue4.Directionsforreadingsamples:TheamountofEBintheexperimentalsampleswillbecalculatedbyinterpolatingtoastandardcurve.Tomakethestandardcurve,performa1:1000dilutionofthe3%stocksolution(30mg/ml)byadding1μlstock:1000μlformamide.Thiswillmakea30μg/mlsolution.Label81.5mltubes1-8foreachofthepointsinthestandardcurve.Thefirstpointonthestandardcurvewillbe12.9μg/ml.Makethisbyadding430μlofthe30μmlsolutionto570μlformamide.Thenperforma2xdilutionseriesbyadding500μlofthe12.9μg/mlsolutionto500μlformamideandrepeateachtimeadding500μlofthemoreconcentratedEBsolutionto500μlformamide.Attheend,thestandardcurvepointswillbe12.9,6.45,3.225,1.6125,0.806,0.403,0.2015,and0.1μg/ml.Bythetimeyougettothelast2dilutions,thesolutionwilllookprettymuchclearbyeye.LoadtheEvansBlueprotocolintheSoftMaxprogram.Inthetemplatewindow,labelthewellsforthe8standardsandalltheexperimentalsamples(usesomesystemsuchasM1duoandM1uttodenotewhichmouseandtissueisineachwell).Add200μI/wellformamideandblanktheplate.Makesuretoremovealloftheformamidefromthecrevicesofthewellbysuctionandadd200μI/wellofeachofthestandardsandexperimentalsamplestoamicrotiterplate.Becarefultoaddthecorrectsolutiontothecorrectwellandtonotcross-contaminate.Readtheplateandprintoutthecurveandtheunknownsgrid.ThenumbersintheMeanResultcolumnaretheamountofEB(inμg/ml).Sincethesampleswereextractedin1ml,thiswillgiveyoutheamount(inμg,oryoucanconverttong)ofEB/tissue.TogettheamountofEBinng/mgtissue,multiplythevaluesintheMeanResultcolumnby1000(toconvertthemtong)anddividethembythewetweightofthetissue.1.Itisdifficulttohandleperfusingmorethan6-8miceinoneday.Thereforeifagreaternumberofmicearetoreceivetreatment,itisbesttostaggerthehormoneinjectionssothatnomorethan6-8aretoreceiveEvansBlueonthefinalday.2Usinganinhaledanestheticispreferredasindictablesuchasavertinseemtostimulatevascularleakageintheintestine.However,themouse"sbreathingmustbecarefullymonitoredandtheamountofisofluraneinhalationadjustedaccordinglytoensurethatitdoesnotdiebeforethe20injectiontimehaselapsed.3Ifmanymice(5-8mice)aregoingtobeperfused,itmaybeeasiestifyoutimetheinjectionsandperfusionstobeoverlapping(i.e.injectonemouseandthenafterwaiting10-15min,injectthenextmousesothatafteryouaredoneperfusingthefirst,thenextmousewillbereadyinafewminutes).Placetheintactmiceoniceuntilyouaredonewiththeperfusionsandthendissectthetissue.4Afterreadingthesamples,the200μlaliquotinthemicrotiterplatecanbediscarded.Storetheremaining800μlinthesolventsavertubesindefinitelyastheycanbere-readonthespectrophotometerinthefutureshouldtheneedarise.

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