FIXATIONWITHFORMALDEHYDE-ACETONE

1.Dulbecco"sPBS,37^oC.

2.Fixationbuffer,i.e.PBS,PHEM,ormodifiedHanks("Cytoskeletonbuffer"),1.33x.

3.Formaldehyde,16%,EMSciences.Storeatroomtemperatureinthefumehood.Markthedatewhenyouopenanewampule.SealtightlywithParafilm.Handlewithcaution.

4.Fresh4%formaldehydesolution.Mix3parts1.33xfixationbufferwith1part16%formaldehyde(e.g.9mland3mlfor1coverslip),inabottledesignatedforfixatives.Captightlyandwarmupinawaterbath.

5.Acetone,chilledto-20^oCinafreezer.Transferinto100mlbeakersdesignatedforfixation,eachcoverslipneedsaseparatebeaker.

6.Forcepsforfixation.

7.Plasticfixationboxes.

1.Transferthewarmfixationsolutionintoaplasticfixationboximmediatelybeforefixation.

2.Drainallthemediumfromthecoverslipdish.Rinseout2timeswithwarmPBS.

3.Quicklyandcarefullyremovethecoverslip.

4.Placeonthesurfaceofthefixationsolution,cellsidefacingdown.Thecoverslipshouldfloatonthesurfaceofthesolution.Rocktheboxintermittently.Fixfor10minatroomtemperature.

5.Removethecoverslipwithapairoffixationforceps.Rinsewithdistilledwater2times.

6.Dipthecoverslipintothecoldacetone.Rockgently.Letsitfor5min.

7.RemovethecoverslipfromacetoneandrinseimmediatelywithPBS.Donotletthesurfacedryout.

8.Proceedwithstaining.

9.Usedacetoneshouldbeemptiedintoadesignatedcontainer.

10.Note:Theacetonestepmaybereplacedbyadding0.1%TritonX-100tosolution4anddofixationandextractioninonestep.