YeastIFwithoutDehydration

DavidAmberg

NotethisprotocolisamodifiedversionoftheprotocolfromMarkRoseintheCSHYeastGeneticsCourseManual.

1. Growcellsattheappropriatetemperatureto5x10E6in5mlsYPD.Add0.5mls37%formaldehyde(bestgrade)andincubateontherolleratsametemp.for10min.
2. Spindowncells2Kx3min.andresUSPendin5mls40mMKPO4pH6.5/500uMMgCl2+0.5mlsformaldehyde.Incubate1hrat30¡C(orprevioustemp).
3. Washthecellstwotimesinthepreviousbuffer(noformaldehyde)andonceinthesamebuffercontaining1.2Msorbitol.Beverygentlewiththecells!Resuspendin0.5mlssorbitolbuffer.Thecellscanbestoredatthispointovernightat4¡C.
4. Zymolyasetreatthecellswith30ul10mg/mlZymolyase(100T)at30¡Cforanywherebetween10-30minormore.Examinecellsonaphasemicroscope.Whencellsaredarkandmishapen,youhavegonewaytoofar,ifbrightandrefractiletheyprobablyneedtobeincubatedlonger.Iftheylookgoodbutaredullgreytheyarejuuuussstttright.Isuggestthisbedoneasatimecourseasitisthesinglemostvariablepartoftheprocedureandunfortunatelyitisthemostcriticalaswell.
5. Washthecellsonceinsorbitolbufferandsuspendin100-500ulofthesame.BEVERYVERYGENTLE!Placeonice.
6. Coatthewellsoftefloncoatedslideswith0.1%polylysine(>400,000MW)inwaterfor10minatRT.Spinthissolutionfor10mininamicrofugeimmedietlybeforeuseandstayawayfromthebottom.Ingeneraldohighspeedspinsofallsolutionsthatgoonthewellsrightbeforetheyareused.Incubatetheslidesinamoistchamber.
7. Washthewells4-5timeswithcleanspunwateranddry.Thesecanbepreparedinadvanceifkeptdustfree.
8. Spot20ulcellsuspensiononthewellsandincubateatRTfor10min.Isuggestyoudoeachsampleinduplicate.Aspirateoffmostoftheliquidbutnotall,donotlettheslidesdry!BlockthecellsinPBSpH7.4/0.5%BSA/0.5%ovalbumin/0.5%Tween20.Highspeedspinthissolution.Beforewarnedthatthetweenreducesthehydrophobicityoftheteflonandsamplemixingcanoccur.Ifindthatifyououtlinethewellswithasharpybeforeapplyingthecells,youcanpreventmixing.Incubatefor15minatRT.Thecellscanbeincubatedforovernightatthisstep.
9. Incubatethecellsinblockcontainingantibodyattheappropriatedilution(determineexperimentally).Adilutionseriesof1:100to1:10,000shouldcoverit.IncubateatRTfor1hrorlonger,dependingontheantibody.Sometimesanovernightincubationishelpful.Jon"santi-actinGuineapigantibodiesfromanimal#1arebestat1:6,000,#2and#3arebestat1:2,000.
10. Washthecells4x5minwiththeblocksolution.Sometimeslongerincubationtimesmaylowerthebackgroundbutthisisusuallysufficient.Donotletthecellsdry!
11. Incubateinsecondaryantibodyconjugatedilutedinblocksolutionfor1hratRT.Youmayneedtodeterminetheconcentrationbestforyoursecondaryantibodyaswell.TheCappelanti-GuineaPig-FITCisbestat1:800-1:1,000.
12. Washasbefore.Aspiratemostoflastwashoffthecellsbutdonotdryandmountimmediatly.Mountbysloppingthemountsolutionovertheslide(nobubbles)andgraduallylayingthecoverslipdownbythelongaxis.Laypapertowelsovertheslideandsqueezeouttheexcessmount.Whileholdingtheslidedownwiththefingersofonehand,cleantheslidewithakimwipe.Finallysealtheedgesoftheslidewithnailpolish.IfindthatSallyHansen"s"HARDASNAILS"worksbest.Allowtodryandstoreat-20¡Cuntilyouarereadytoviewtheresults.Beforeyouplacetheslideonthescope,completelycleananyresidualmountfromtheslide,thisstuffisbadfortheobjectives.

Reagents

Zymolyase

Usethegoodstuff100T.Dissolveinthesorbitolcontainingphosphatebufferdescribedintheprotocol,spinfor10mininamicrofuge,removetoafreshtubeandquickfreezeonliquidnitrogen.Storeat-80¡C.Donotrepeatedlyfreezeandthaw,ifyoudoalwaysquickfreeze.

Polylysine

Buythegoodstufffromsigma,>400,000MW.Dissolveasa1%stocksolutioningoodwater.Quickfreezeinaliqoutesandstoreat-80¡C.Sameappliesasforzymolyase.

DAPI

Stocksolutionshouldbeat1mg/mlinwaterandstoredat-20¡C.

Mount

Dissolve100mgp-phenylenediaminein10mlsPBS,adjustthepHtoabove8.0with0.5MNaCarbonatebuffer(pH9.0)andbringthevolumeto100mlswithglycerol.AddDapito50ng/ml.Mixthoroughlyandstoreat-20¡C.Itturnsbrownwhenitisbad.