EnzymeImmunostainingforBrdU:

1. WashfrozensectionswithPBS2x.
2. Putthemin0.1%Pepsinin0.1NHCL(inPBS)at37�Cfor50min.
3. Thenin0.3%hydrogenperoxideinPBS,30min.ThenrinsewithPBS3x.
4. Thenin5%normalgoatseruminPBSwith1%BSAatRTfor1hour.
5. Mouseanti-BrdUdilutedto1:1000with1%NGSand1%BSAinPBS,incubateat4�Covernight.
6. RinsewithPBS3x.
7. GoatBiotinylatedanti-MouseIgG1:200in1%NGS,1%BSAinPBS,incubateatRTfor1hour.RinseinPBS3x.
8. IncubatewithABCreagentinPBScontaining0.1%Tween20atRTfor1hour..(ABCreagentsolution:5mlPBS/0.1%Tween20with1dropAand1dropB,mixwell).
9. RinsewithPBS3x.
10. DABreaction:2.5mlofdistilledwater+1dropBuffer+2dropDABsolution+1drophydrogenperoxide.~Needsabout8mintostopDABreaction.
11. Rinsewithdistilledwater3x.Counterstainandmount.

CultureCellFluorescenceImmunostainingforBrdU:

1. WashplateswithPBS3x.
2. Fixthemin4%PFA,20minatRT.
3. WashtheminPBS3x.
4. 0.5%TritonX-100inPBS,15minat4�C
5. WashslidesinPBS3x.
6. 2NHCL(inDistilledwater)30minat37�C(DNAdenaturation).
7. WashinPBS5x
8. Incubateslideswith1%BSAinPBSatRTfor30min.
9. Mouseanti-BrdUdilutedto1:1000with1%NDSand1%BSAinPBS,incubateatRTfor2hrs.
10. WashwithPBS3x.
11. 1:100dilutionofRhodamine(Tritc)conjugatedDonkeyantimouseIgGwith1%NDS,1%BSAinPBS,incubateatRTfor1hr.
12. WashinPBS3x
13. Mountwith50%glycerolinPBS.

CultureCellEnzymeImmunostainingforBrdU:

1. WashplateswithinPBS3x.
2. Put4%PFA20minatRT.
3. WashwithPBS3x.
4. 0.5%TritonX-100inPBS,15minat4�C
5. WashwithPBS3x.
6. Incubatewith2NHCL(inDistilledwater)30minat37�C(DNAdenaturation).
7. WashwithPBS5x
8. 1%BSA,incubatefor30minatRT.
9. Mouseanti-BrdUdilutedto1:1000with1%NGS,1%BSAinPBS,incubateatRTfor2hrs.
10. WashwithPBS3x.
11. DiluteGoatBionytilatedanti-MouseIgGto1:200with1%NGSand0.1%BSAinPBS,incubateatRTfor1hour.RinsewithPBS3x.
12. ABCatRTfor1hour.RinseinPBS3x.(ABCsolution:5mlPBSwith1dropAand1dropB,mixwell)
13. DABreaction:2.5mlofdistilledwater+1dropBuffer+2dropDABsolution+1drophydrogenperoxide.Needsabout8mintostopDABreaction.
14. Rinsewithdistilledwater3x.
15. CountstainwithMayer’sHematoxylin

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