Bromodeoxyuridine Staining of Mammary Tissue相关交流-蚂蚁淘在线

Bromodeoxyuridine Staining of Mammary Tissue

蚂蚁淘 /发布时间:1630776610 /浏览量:306

ThefollowingisaprocedurewehaveusedandmodifiedfordetectingDNAsynthesisincellsofmousemammaryglands

I.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.

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Bromodeoxyuridine&nbsp;Staining&nbsp;of&nbsp;Mammary&nbsp;Tissue

I.BrdUTreatment

II.BrdUDetection

A.Perfusionfixed,paraffinsections:

Forfreshfrozen,cryostatsections:

III.SolutionsforBrdUStaining

Bromodeoxyuridine Staining of Mammary Tissue