Materials:? 1)? 1.0M? KPO4? pH? 6.5? :??? Make? by? mixing? 33? mls? of? 1M? K2HPO4? with67 mls of 1M KH2PO4? 2)? 0.1M? KPO4 pH 6.5? 3)? 0.1M? KPO4? pH? 7.5:? Make? 500? mls? by? mixing? 41.7? mls? of? 1MK2HPO4? and? 8.3? mls? of? 1M? KH2PO4? with? 450? mls? of? ddH2O? 4)? 37%? Formaldehyde? 5)? KS? Buffer:? 0.1? M? KPO4? pH? 7.5/1.2? M? Sorbitol? 6)? Zymolyase? 100T:??? 5? mg/ml? solution? in? 0.1M? KPO4/0.5%? β-mercaptoethanol? 7)? β-mercaptoethanol? 8)? HS? Buffer:? 0.1? M? Hepes? pH? 7.4/1.0? M? Sorbitol? 9)? HS/SDS? Buffer:? 0.1? M? Hepes? pH? 7.4/1.0? M? Sorbitol/0.5%? SDS10)? 0.1? %? Polylysine? (MW? >300,000;? Sigma? Cat.? No.? P-1524)? Storedin? 1ml? or? 10? ml? aliquots? at? -20°C.??? After? thawing? microfugealiquot? for? 5? min.? at? 4°C? before? adding? to? slides.11) Multiwell? teflon? masked? slides? (Carlson? Scientific? Inc.? PeotoneIL.? Cat.? No.100806)12) PBT? Buffer:? PBS? with1? mg/ml? BSA? and? 0.02%? Tween? 2013) PrimaryAntibodies:? Affinity? purified? polyclonal? antibodies? or?????????????????????????????????monoclonal? antibodies? from? cell? culture? supernatant? (notfrom????ascites? fluid).??? Dilutions? must? be? determined? emperically? for? eachprimary? but? common? ranges? are? for? affinity? purified? sera? 1:10to? 1:100? in? PBT? Buffer.? For? cell? culture? supernatants? 1:1? to? 1:5dilutions? are? normally? used? (if? cell? supernatants? wereconcentrated? by? ammonium? sulfate? precipitation? then? 1:10? to1:50dilutions? are? often? used).??? After? diluting? in? PBT? buffer? spinsecondary? in? microfuge? (at? 4°C)? for? 5? min? to? pellet? any? debris.14) SecondaryAntibodies:? Jackson? Immunoresearch? Laboratories?????????????????????????????????????makes? high? quality? secondary? antibodies? which? are? good? forboth? single? and? double? labeling? experiments.? We've? been? using:Texas? Red? conjugated? AffiniPure? Donkey? Anti-Mouse? IgG(Cat#715-075-141)? and? Fluorescein? (DTAF)? conjugatedAffiniPure? Donkey? Anti-Rabbit? IgG? (Cat#711-015-132).??? Theyare? sent? as? freeze-dried? powder? which? should? be? reconstitutedwith? ddH2O? according? to? directions? and? then? aliquoted? into? 50μl/tube? (marked? with? name? of? Ab? and? Date)? and? frozen? on? dry???????ice? and? stored? in? the? "Secondary? Antibodies"? Box? in? the? -80°CFreezer.? After? thawing? an? aliquot? keep? it? at? 4°C--it? should? begood? for? about? a? month.??? These? secondary? Abs? work? very? wellfor? both? double? and? single? labeling? and? should? be? used? at? 1:50to? 1:100? dilutions? each? (in? PBT? buffer).? After? diluting? in? PBTthe? solution? should? be? microfuged? for? 5? min? at? 4°C? to? removeany? precipitates? that? may? have? formed.15) Mounting? Medium:??? Dissolve? 10? mg? of? ρ-phenylenediamine(Sigma? Cat.? No.? P-6001)? in? 1? ml? of? 1M? K2HP04 in? a? 1.5? mleppendorf? tube.??? Vortex? vigorously? for? several? minutes,? coverwith? foil,? and? rock? on? nutator? for? 20-30? minutes.??? Vortex? againand? microfuge? for? 5? min.??? Remove? the? top? 800? μl? to? a? 15? mltube.??? Add? 7.2? ml? of? glycerol? and? mix? by? vortexing.??? Divide? into500? μl? aliquots? and? store? at? -80°C? (in? the? "Secondary? Ab"? Box).??Optional:? For? nuclear? staining? DAPI? can? be? added? to? mountingmedium? as? follows:??? make? a? 1? mg/ml? DAPI? (4',6-diamidino-2-phenylindole? dihydrochloride)? solution? in? water,? dilute? it? 1:100with? water? and? add? 2.25? μl? to? 1? ml? of? mounting? media.Method:1)??? For? each? strain? to? be? examined? inoculate? 50? mls? of? YPD? or? SD(with? the? appropriate? amino? acid? supplements).? Grow? overnight? at25°C? until? culture? is? at? a? OD599? between? 0.5? and? 0.8.??? For? examingunshifted? cells? proceed? directly? to? Step? 2.??? For? examining? sec7ts andsec6ts? strains? spin? cells? out? of? YPD? at? OD599? of? 0.5-0.6? and? resuspendin? the? same? volume? of? YP? with? 0.2%? Glucose? and? then? incubate? in? a37°C? shaking? water? bath? for? 2? hrs.? Check? OD599? and? then? cool? for? 5min? on? ice? until? the? culture? is? at? room? temperature? before? fixing.2 ) Fix? yeast? directly? in? the? culture? by? adding? to? each? 50? mls? ofculture:? 5? mls? of? 1.0? M? KPO4 pH? 6.5? and? 6? mls? of? 37%? Formaldehydedirectly? to? the? culture? flask.??? Incubate? at? room? temperature? withgentle? shaking? for? 30? min.??? Transfer? an? amount? of? cells? that? is? equalto? about? 5-10? OD599? units? for? each? culture? into? a? 50? ml? centrifugetube? and? spin? in? the? Beckman? table-top? centrifuge? for? 5? min.? at? 2000RPM? at? 25°C.??? Aspirate? off? the? supernatant? and? resuspend? each? pelletin? 5? mls? of? 0.1? M? KPO4? pH? 6.5? and? transfer? to? a? 15? ml? centrifugetube.??? Add? 0.6? mls? of? formaldehyde? to? each? tube? and? incubate? atroom? temperature? for? 1.5? hrs? with? gentle? rocking.3)??? Harvest? fixed? cells? by? centrifuging? at? 2.2K? RPM? for? 5? min.? at25°C.??? Aspirate? off? supernatant? and? resuspend? in? 5? mls? of? 0.1? M? KPO4pH? 7.5? and? repeat? spin.??? Aspirate? off? sup.? and? resuspend? cells? in? 5mls? of? 0.1? M? KPO4/1.2? M? sorbitol.??? Fixed? cells? can? now? be? storedovernight? (or? for? several? days)? at? 4°C.4 ) Prepare? Zymolyase? solution? by? dissolving? 2-5? mg? of? Zymolyasein? 0.1? M? KPO4? pH? 7.5? to? give? 5? mg/ml? solution.? Add? β-mercaptoethanol? to? 0.5? %? (i.e.? 5? μl/ml),? mix? by? gently? vortexing? andlet? sit? at? room? temp.? for? 20-30? min? to? dissolve.5)? Harvest? fixed? cells? at? 2.2K? RPM? for? 5? min.??? Aspirate? offsupernatant? and? resuspend? in? 1? ml? of? 0.1? M? KPO4? pH? 7.5/1.2? MSorbitol.? Transfer? cells? to? a? 13x100? mm? glass? culture? tube? (i.e.? bluecapped? tubes).? Add? 45? μl? of? 5? mg/ml? Zymolyase? solution? and? 5? μl? ofβ-mercaptoethanol? and? incubate? at? 30°C? for? 30? min? with? occassionalmixing? by? finger-flicking.6)? Harvest? spheroplasts? at? 2.2K? RPM? for? 5? min? at? 25°C.??? Resuspendcells? in? 3? mls? of? HS? Buffer? and? spin? at? 2.2K? RPM.??? Aspirate? offsupernatant? and? repeat? wash? one? time.7 ) To? permeabilize? cells? resuspend? spheroplasts? in? 3? mls? ofHS/SDS? and? incubate? at? room? temperature? for? 5? min.??? Centrifuge? at2.2K? RPM? for? 5? min? and? resuspend? cells? in? 3? mls? of? HS? Buffer? andspin? at? 2.2K? RPM.??? Aspirate? supernatant? and? repeat? wash? with? HSBuffer.??? Resuspend? cells? in? 1? ml? of? HS? Buffer.??? Do? not? store? the? cellsfor? more? than? a? few? hours? at? this? stage.8 ) Set? up? an? aspirator? with? "pulled"? pasteur? pipette? at? the? endnear? to? where? the? slides? are? to? be? processed? to? speed? up? the? washingprocedures.??? Prepare? slides? by? adding? 20? μl? of? 0.1%? polylysine? toeach? well? and? incubate? for? 5-10? min.? Aspirate? off? and? wash? each? well3? times? with? one? drop? of? ddH2O.9)? Place? 20? μl? of? spheroplasted/permeabilized? cell? suspension? oneach? well.??? Incubate? for? 5-10? min.??? Aspirate? each? well? and? add? onedrop? per? well? of? PBT? Buffer.? Repeat? twice? and? let? sit? while? primaryAb? dilutions? are? prepared.10) Prepare? dilutions? of? primary? antibodies? in? PBT? and? microfugebefore? use.? Add? 20? μl? of? diluted? primary? antibody? and? transfer? slidesto? a? plastic? box? with? a? moistened? paper? towel? at? the? bottom.Incubate? 60-90? min.11)??? Carefully? remove? slides? from? box? to? benchtop? for? washing.??? Towash? slides? rapidly,? hold? aspirating? pipette? in? one? hand? and? anotherpastuer? pipette? with? a? rubber? bulb? containing? PBT? Buffer? in? theother? hand.??? For? each? well? aspirate? the? liquid? and? then? add? one? dropof? PBT,? aspirate? add? PBT,? repeat? this? twice? (leaving? the? well? with? adrop? of? PBT)? and? then? move? on? to? the? next? well.??? It? is? important? thatthe? wells? do? not? dry? out? once? antibodies? have? been? added.??? Once? allthe? wells? have? been? washed? move? on? the? next? slide.??? Then? repeatthis? entire? procedure? twice? so? that? each? well? has? been? washed? atleast? nine-times.??? This? may? seem? excessive? but? it? often? dramaticallyincreases? the? quality? of? the? images? obtained? and? once? you? get? thehang? of? using? both? hands? it? can? be? done? fairly? quickly.12) Wrap? plastic? box? used? for? primary? Ab? incubations? in? foil? so? it? islight? tight.??? Place? slides? inside? and? place? 20? μl? of? secondary? antibodyin? each? well.??? Incubate? for? 60-90? min? at? room? temperature.13) Wash? the? wells? as? before? with? a? total? of? at? least? 9-10? washes? ofPBT? Buffer.??? Aspirate? the? wells? dry? after? the? last? wash? and? let? air? dryunderneath? a? piece? of? foil? (make? a? tent)? for? 10-15? min.14) Place? 4? evenly? spaced? small? drops? of? mounting? medium? inbetween? the? two? columns? of? wells? in? the? middle? of? the? slide.Carefully? place? the? coverslip? over? the? slide? and? let? mounting? mediumspread.? Seal? the? slides? with? nailpolish,? let? dry? 10-15? min.??? Then? rinseoff? slides? with? ddH20,? dry? with? a? kimwipe? and? view? immediately? orstore? in? dark? at? -20°C.For? More? information? and? alternative? methods? see:"Immunoflourescence? Methods? for? Yeast"? by? Pringle? et.al? on? pg.? 565in? GuidetoYeastGenetics? andMolecularBiology? edited? by? Guthrie????????????????????????????????????????????????????????????????????????????and? Fink? (Vol.? 194? of? Methods? in? Enzymology).

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