BrdUImmunoperoxidase Staining of Paraffin Tissue Sections

Syringes,1mLwith36GneedlesBrdU:3-10mg/mLinPBSFixative:MethylCarnoy"s,or4%paraformaldehydeinPBSParaffinprocessingmaterialsOrganicsolvents:Xylene,EtOH,MeOH0.3%H2O2:2mL30%H2O2in200mLMeOHTrypsin:Dilute(10xstock,-20ºC)1/5inPBS2.5NHCl:DiluteconcentratedHCl1/22(v/v)inH2OMouseanti-BrDU:DAKOcat#M0744,$205/mLBiotinylatedhorseanti-mouseIgG,Avidin,biotin-HRP,Horseserum:Vector,VectastainABCkit,#PK4002$185Colorsubstrate,(makefresh):25mL50mMTrispH8,1mLDAB(3,3-diaminobenzidine,25mg/mLstock,-20ºC),100µLNiCl2(10%w/vstock,4ºC),10µL30%H2O2.Counterstains:MethylGreenordiluteEosin(1/4xin80%EtOH)

BrdULabeling1.Inject0.1mLofBrdUpergmofbodyweight(30-100mg/kg).Usehigherdosesforshortlabelingtimes.Labelfor1-2hourstomeasurethenumberofcellsinS-phase.MaximalstainingintensitywilloccurrifthelabelingtimeexceedsthedurationofS-phase(6hours)Longerlabeling(e.g.1-7days)doesnotgivethenumberofcellsinS-phaseatagiventimepointbutwillgivetheproliferativefractionforthattimeperiod.Tolabellongerthan12hoursgiverepeateddosesevery24hours.UselowerBrdUdose(30mg/kg)forprolongedlabellingtoavoidchemotherapy-liketoxcity.LowerdosesofBrdUalsoeffectstheintensityofstainingandindirectlythenumberofcellsthatwillbedeemed"positive".

2.Control:ThebestnegativecontrolisanadditionalanimalgivennoBrdU(ormoreformally-injectedwithPBSonly).Thisisa"noantigen"controlandissuperiortousing"noprimaryantibody"asanegativecontroltoaccuratelyjudgethelevelofbackgroundstaining.Usesmallintestineasapositivecontrolsincethisgivesrobustlabelingintheepithelialcryptsevenwithshortlabelingtimes.

TissuePreparation1.PlacefreshlydissectedtissuesintissuecassettesandfixeitherinMethylCarnoy"sfor1hourorparaformaldehydeovernight.2.Embedinparaffin,andcut4µMsectionsonglassslides(seeTissueProcessingprotocol).

ImmunostainingInPlasticTubs(200mL):3)Deparaffinize:Xylene2"(x3)100%EtOH2"(x3)70%EtOH2"(x1)4)Quenchendogenousperoxidaseactivity(optional):MeOH2"(x1)0.3%H2O2inMeOH20"(x1)5)PBS5"(x3).

InCoplinjars(25mL):

6)Todecreaseantigenmaskingbychromatinproteinsdigestin2XTrypsin(1mg/mLinPBS)10"(x1)

andthenwashinPBS2"(x3)

7)Denature(depurinate)theDNAwith2.5NHCl,37°C15"(x1),washinPBS2"(x3).(0.1NNaOHcanalsobeusedbutmaybeharshoncertaintissues).Steps6and7mayneedtobealterediftheydestroyotherantigensnecessaryfordoubleimmunostaining.

InHumidChambers(ateachstepuse100µLandcoverwithglassslips.ToremovetheslipsdiptheslideinajarwithPBS.Don"tpullofftheslipsasthiswillscratchthespecimen):

8)BlockwithHorseserum(1/80inPBS)30"(x1).WashinPBS2"(x3)

9)Primaryantibody100µL(1/200mousea-BrDU)(r.t.or37ºC)1hr(x1).Wash(x3).

10)Secondaryantibody(biotinylatedhorseanti-mouseIgG)1/250,30"(x1).Wash(x3)

11)100µLAvidin+Biotin-HRPmix(1/125v/vSol.A,1/125SolB),30"(x1).Wash(x3)

InCoplinjars(25mL):

12)50mMTrispH837°C(x1)

DAB/NiCl2Colorsubstrate37°C,3"(x1).

RinseunderH₂Otapfor5minutes.

InPlastictubs(200mL):

13)Counterstaininmethylgreenfor20"ordiluteEosin(1/4xinEtOH)for2".

14)Dehydrateingradedethanol:

95%EtOH1"(x2),

100%EtOH1"(x2),

HistoclearorXylene1"(x2)(Methylgreenisnotstableinxylene)

15)Add1-2dropsofPermountwithaglassrodandcoverwithglassslip.Cureovernightinfumehood.Don"tstackslidesontopofeachotheruntilfullycured(2days).