LEVELII

Materials

• Coverslipculturesofanappropriatemonolayercellline
• Phosphatebufferedsaline(PBS)
• Acetone/Methanol(absolute)ina50:50volumemixture
• Rabbitanti-tubulin(orotherprimaryantibodytotubulin)
• FITC-labeledgoatanti-rabbit(orsecondaryantibodytomatchtheprimary)
• FluorescenceMountingMedia
• FluorescentMicroscopeequippedwith490nmexcitationfilterand515nmbarrierfilter
• Kodachromefilmorequivalentcolorslidefilm(KodakTri-XorIlfordHP400maybesubstitutedforblackandwhitephotography)

Procedure

1. Setupacoverslipcultureofanappropriatecellline24hourspriortothelab.Thisisbestaccomplishedbydrysterilizationof#1coverslipswhicharesubsequentlyplacedinplastictissuecultureplates.Cellsareplacedonthecoverslipswithsufficientmediatocoverandallowedtogrowfor24hours.Thereshouldbesufficientcellstoviewcomfortably,buttheyshouldnotbecrowdedontheslide.

2. Removethecoverslipfromthecultureplateanddipseveraltimesinabeakerofphosphatebufferedsaline(PBS)torinseofftheculturemedia.Drain,butdonotallowtodry.

3. Immediatelyimmerseina50:50mixtureofacetone/methanolatroomtemperature.Allowthecoverslipstoremainintheacetone/methanolfor2minutes.

4. Removethecoverslipsfromtheacetonemixtureandrinse2XwithPBS.

5. Preparea1/40anti-tubulindilutionusingPBS.PbSalonemaybeusedorbetter,augmentthePBSwith3%(w/v)BovineSerumAlbumin(BSA).

   Itmaybenecessarytochecktheappropriateantibodydilution.Ifso,make1/10,1/100,1/1,000and1/10,000dilutionstoestablishthecorrecttiter.Workingdilutionsalsomayvarywiththemanufacturer-checktheliteraturethataccompaniesyourprimaryantibody.

6. PlacethecoverslipinapetriplatecontainingfilterpapermoistenedwithPBS.Makesurethecellsarepointedupwhenplacedinthepetriplate!Floodthecoverslipwith50mlof1/40dilutionoftheprimaryantibody(orasdeterminedinstep5).

7. Incubateatroomtemperaturefor1-4hours.Theincubationmaybeleftovernightifnecessary.

8. Wash3XwithPBS.PlacecoverslipsinanewpetriplatecontainingPBSmoistenedfilterpaper.

9. Apply50mlofFITC-labeledsecondantibody.A1/100dilutionusuallyissatisfactory.Youmayneedtodeterminetheappropriatedilutionbasedonmanufacturerdirectionsorthroughtrialanderrordilutionsintherangeof1/10to1/300.

10. Incubatefor30minutesatroomtemperature.

11. Wash3XwithPBS.

12. Placeadropofglycerinorappropriatecommercialfluorescentmountingmediaonaslideandplacethecoverslipontotheslidewiththecellsfacingdownintotheglycerin.

13. Observeimmediatelywithafluorescentmicroscopeadjustedforfluorescein(490nmexcitationand515barrierfilter).TheslidesarebestphotographedusingKodakEktachromeorequivalentwithanASAor200-400.Anexposureof1-2secondsisusuallysufficient,althoughforlowlight,30secondsmayberequired.Itisbesttomakeatestexposurerollifaphotometerisnotavailable.

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