ThefollowingisaprocedurewehaveusedandmodifiedfordetectingDNAsynthesisincellsofmousemammaryglandsI.BrdUTreatment
Micewereinjectedi.p.with0.01mL/gbromodeoxyuridine(AmershamCat.#RPN201).Animalsweresacrificedtwohourslaterandperfusionfixedwith4%paraformaldehyde/PBS.Mammaryglandswereremoved,postfixedin4%paraformaldehydeovernightthenparaffinembeddedandsectionedandmountedontoslides.Alternatively,miceweresacrificedandtheglandsremovedimmediatelywithoutfixationandfrozenforcryostatsectioning.Notsurprisingly,themorphologyisbestontheparaffinsections.(AttachedphotomicrographshowsalabeledductintheaBDominalglandfromapost-pubertalmouse.)II.BrdUDetection
A.Perfusionfixed,paraffinsections:
1.Deparaffinizesectionsusingstandardxylene/alcoholprocedures.2.Treatsectionswith0.1%Trypsinin0.1MTBS(pH7.5)+0.1%CaCl2for20minutesat37oCinahumidbox.3.Rinse2x3mininPBS.4.Treatsectionswith0.3%hydrogenperoxidefor10minutesatroomtemperature.5.Rinse2x3mininPBS.6.Treatwith2NHClfor1hourat37oC.7.Rinse2x5minin0.1MboratebufferpH8.5.8.Rinse2x3mininPBS.7.Treatwith0.2%TritonX-100for30minutesatroomtemperature.8.Rinse3x5mininPBS.9.Blocksections1-2hoursatroomtemperaturewithPGBSuperblock(seebelow)inhumidchamber.10.RemoveSuperblockandincubatesectionsovernightat4oCwithanti-BUdR-PODantibody(BoehringerMannheimCat.#1585860)diluted1:15inPGBdiluent.11.Rinsesections3x5mininPBS.12.IncubatewithunactivatedDABfor5minutesfollowedbyactivatedDABfor10minutesatroomtemperatureinthedark.13.Rinsesectionswithdistilledwaterandcoverslipwithanaqueousmountingmedia.Forfreshfrozen,cryostatsections:
Takeslidesfrom-70andimmediatelypostfixfor10minutesin4%paraformaldehyde.RinseinseveralchangesofPBSandprocedewiththeaboveprocedurebeginningwithstep4.Note:thetrypsintreatmentwilltearupyourtissueandisnotnecessary.Also,reductionoftheHCltreatementto30minissufficientforgoodstainingandwillleavethesectionsinbettershape.III.SolutionsforBrdUStaining
PGBSuperblockfor100mLFinalConc.BSA10g10%NaN30.5mLof10%0.05%NormalGoatSerum10mL10%0.5MPO4(pH7.6)79.5mLPGBDiluentfor50mLPGBSuperblock10mLTritonX-100100mLPBS40mLDABSolutionDABStock-3"-3"diaminobenzADIne,2.5mg/mLin0.1MPO4,pH7.6.UnactivatedDAB-1mLDAB+19mL0.1MPO4+450mL1%aq.NiCl2,pH7.6.ActivatedDAB-10mLunactivatedDAB+23mL30%H2O2.
