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whole mount preparations

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imageofawholemountfroma4wkoldvirgin

Protocol

SpreadtissueonglassslideFixinCarnoy"sfixativefor2to4hoursatr.t.Washin70%EtOHfor15minChangegraduallytodistilledwaterRinseindistilledwaterfor5minStainincarminealumO/NWashin70%EtOH15minWashin95%EtOH15minWashin100%EtOH15minClearinxyleneandmountwithPermount

Afterphotographicdocumentationthetissuecanbeembeddedinparaffinforsectioningandconventionalhistologicalstaining.

ImmerseslideintoxylenetoremovemountingmediumChangexylene2timesTransferto1:1xylene:paraffin(60°C)Changexylene2timesandembedinparaffin

Carnoy"sFix:6parts100%EtOH3partsCHCl31partglacialaceticacid

CarmineAlumStain:Place1gcarmine(SigmaC1022)and2.5galuminumpotassiumsulfate(SigmaA7167)in500mldistilledwaterandboilfor20min.Adjustfinalvolumeto500mlwithwater.Filterandaddacrystalofthymolaspreservative.Refrigerate.Canbeusedforseveralmonths.Discardwhencolorbecomesweak.

SummaryThebacterialbeta-galactosidasegenelacZisfrequentlyusedasareportergene.TheexpressionoftransgenicconstructscanbemonitoredbyhistochemistrywiththechromogenicsubstrateX-gal.Thisallowsprecisecellularlocalizationofgeneactivity.Undercertainconditions(seebelow),nobackgroundstainingisdetectableinmammarytissue.Beta-galactosidaseactivitycanbeassayedinsmallpiecesoftissueoroncryosections.
Method1.Spreadglandtissueonapieceofpaperandfixfor1-2hrsin2%paraformaldehyde,0.25%glutaraldehyde,0.01%NP-40inPBS(use10to20mlinascintillationvial)2.RinseinPBS,removefrompaper3.Add10mlofPBSwith2mMMgCl2,0,01%Na-deoxycholate,0.02%NP-40androckfor2hrs4.Add10mlofX-galstainingbufferwith1mg/mlX-gal(make40mg/mlstockinDMF,storeat-20OC;don"tuseifdiscolored).Incubateat30OCfor24-48hrs,orlessifexpressionisstrong5.Clearinacetone,rehydrateandstainincarminealumO/N.6.Dehydrate,clearinxyleneandmountwithPermount
Alternative:6.Fixagainin4%PFA,dehydrate,embedinparaffinandsection
Stainingbuffer:30mMK4Fe(CN)6[4.983g/500ml]30mMK3Fe(CN)6.3H2O[6.336g/500ml]2mMMgCl2[1ml1M/500ml]0.01%Na-desoxycholate[50mg/500ml]0.02%NP-40[100microl/500ml]1xPBS[50ml10x/500ml]
CommentsFixationtimeiscritical.Overfixationinhibitsenzymeactivity.Closecontrolofreactiontemperature(30OC)andpH(PBS7.2)arecriticalforeliminationofendogenousbeta-galactosidaseactivity.IncasesofhighenzymeactivitytheX-galproductformsaprecipitateonthesurfaceofthetissuewhichpreventspenetrationofsubstrateintodeeperregions.Whensuchtissuesaresectionedonlytheouterlayersofcellswillshowstaining.Toevalutetransgeneexpressioninthecenterofthetissueitwillbenecessarytoperformthestainingprocedureonfrozensections.
imageofasectionfrommammarytissueofaWAP-lacZmouseatday10oflactation(seeRobinsonetal.,1995;1996).
1.EmbedtissueinOTCandfreeze.Optionally,tissuescanbefixedbeforefreezing.Thismakesiteasiertosectionbutmayreducesensitivity.2.Prepare10-20micronsectionsongelatine-coatedslides,letairdry.3.Fixsections5minin2%paraformaldehyde,0.125%glutaraldehydeinPBS.(Omitthisstepiftissuesarefixedbeforefreezing)4.Aspiratefixativeandincubateslides3x1mininPBSwith2mMMgCl2.5.Incubate3x2mininPBS,MgCl2,NP40,desoxycholate.6.Incubate2minwithstainingbufferwithoutx-gal.7.Addx-galtostainingbufferandincubateat30or37OC.Severalhourstoovernight.
Allincubationsareperformedinamoistchamber.

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whole&nbsp;mount&nbsp;preparations

Protocol

whole mount preparations