RhodaminePhalloidin/Calcofluor Staining
来自 : 蚂蚁淘
Rhodamine-Phalloidin/CalcofluorStaining
DavidAmberg
- Grow50mlsofyeastto5x10E6.
- Addformadehydetothemediato4%(33mls.of10%).Fixinmediaattemperaturefor10min.
- Spindowncells2-3Kfor5min.
- FixcellsinPBScontaining4%formadehydefor1hrattemperature.
- Washcells2timeswithPBSandreconstitutein500ulPBS.
- Remove100ulcellsandadd10ulRhodaminePhalloidin(6.6uMinMeOH)andadd10ul1mg/mlCalcufluor.Incubateinthedarkfor1hr.
- Washthecells5timesin1mlPBS.
- SUSPendthecellsin1dropofimmunofluorescencemountingsolutionandstoreat4¡C(goodforafewdays).
- Visualizebyplacing2ulofcellsuspensionunderastandardsizecoverslipandwaitingforcapilaryactiontodrawtheliquidoutthusflatteningthecells.
Reagents
- Formaldehyde
- polysciences,10%EMGradeCat#04018
- 10XPBS
- 80gramsNaCl+2gramsKCl+14.4gramsNa2HPO4+2.4gramsKH2PO4.pHupondilutionto1Xshouldbe7.2.
- RhodaminePhalloidin
- MolecularprobesInc.,Dissolvedinmethanolaccordingtomanufacturersinstructionsto~6.6uMandstoreddessicatedat-20¡C.Cat#R-415.
- Calcofluor
- akafluorescentbrightener28.SigmaCat#F-6259.Dissolveto1mg/mlinH2Oovernightandstoreat4¡Cinthedark.
- Mount
- Dissolve100mgp-phenylenediaminein10mlsPBS,adjustthepHtoabove8.0with0.5MNaCarbonatebuffer(pH9.0)andbringthevolumeto100mlswithglycerol.AddDapito50ng/ml.Mixthoroughlyandstoreat-20¡C.Itturnsbrownwhenitisbad.
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