Rhodamine-Phalloidin/CalcofluorStaining

DavidAmberg

1. Grow50mlsofyeastto5x10E6.
2. Addformadehydetothemediato4%(33mls.of10%).Fixinmediaattemperaturefor10min.
3. Spindowncells2-3Kfor5min.
4. FixcellsinPBScontaining4%formadehydefor1hrattemperature.
5. Washcells2timeswithPBSandreconstitutein500ulPBS.
6. Remove100ulcellsandadd10ulRhodaminePhalloidin(6.6uMinMeOH)andadd10ul1mg/mlCalcufluor.Incubateinthedarkfor1hr.
7. Washthecells5timesin1mlPBS.
8. SUSPendthecellsin1dropofimmunofluorescencemountingsolutionandstoreat4¡C(goodforafewdays).
9. 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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