EMFixationofEmbryos

AdaptedfollowingadvicefromAndyFireandJimPriess

1.MountembryosonslidesasifforDIC,exceptagarhasfixative.

AgaroseFixative

3ml2%Agarose:1.5%LGTAgarose+.05%HGTAgarose

5ul1MMgCl2

400ul0.2MNaCacodylate(pH7.4)

400ulsucrose(68.46g/100ml)

500ul25%Glutaraldehyde

Makeupabovemixtureimmediatelybeforemountingembryos.Thepadshouldbethick,sousetriplethicknesstapewhenpreparingslide.Transfertheeggstopadswithminimalfluid(topreserveosmolarity).TransferwithapickormouthPipette.Useaneyebrowhairtopushtheeggstothecenterandarrangethemsothattheyareclosetoeachotherwithouttouching.

2.Overlayasmallamountofslightlycooledagarosemixonslideandplaceacoverslip[#1thickness]ontop.Ifoundthatan18mmcoverslipwasbestforScottEmmon"slaser,butthatthesmall12mmcoverslipsworkedbestonAndyFire"slaser.

3.Makeamapoftheembryos-theirarrangementonthepad,theirorientation,andtheirrelativestages.Mountabunchof1,2,and4cellembryosonapad,thenwatchthemgothroughlandmarkevents,likegastrulation,themigrationofthedorsalhypnuclei,orcelldeathstostagethem.

4.Whenanembryoreachesthestageyouwant,permeABIlizetheeggshellandvitellinemembranewithalaser.Shootforedgeofshell.Youcanseethebreakintheshellandthemembrane.Thelaserneedstobeatfullpower.OnAndy"s[Olympus]scope,weusea40xoilobjective.Alsokill(explode)anyembryosthatyoudon"twantandrecordtheirpositionsonyour"map".Useafewembryostopretestthefixsolutionforosmolarity[seebelow].

5.Placetheslideinahumidifiedchamberatroomtemperaturefor2hourstoallowfixation.

6.Removethecoverslipbypipetting25mMNaHEPES(pH7.5)aroundthebaseofthepad,thengentlyslidethecoverslipoff.Eggswillusallystayinplace.

7.Begindehydrationsandwashes.Eitherleavethegroupintheiroriginalagarpads,orcutupthepadandplaceindivdualeggsinseparatewellsofa9-wellglassplate.Eitherway,trimtheagarosewithacleannewrazorblade.

8.RinseinNaHEPES:

2X30"

1X2"

9.Washin0.2MNaCacodylatepH7.4

2X30"

1X5"

1x10"

2X1hr

10.Post-Fixin1%OsO4+1%KFe(CN)6in.2MNaCac-1to2hours.

11.Washin0.1MNaCac

2X30"

1X5"

2X10"

12.Post-Fixin0.2%tannicacidin0.1MCac-15"

13.Washin0.1MNaCac

2X30"

1X5"

2X10"

Washin0.1MNaAcetate

2X30"

1X5"

1X10"

1X30"

14.Stain1%UranylAcetatein0.1MNaAcetate-3hours.

15.WashininNaAcetate

2X30"

2X10"

Washin0.1MHEPES

1X30"

2X5"

16.Beforethedehydrationseries,remounttheembryosintolargechunks(~1cc)ofagar(2%)andthencuteachblockintoadistinctshapesothateveryembryocanbedistinguishedfromalltheotherembryos.Thelargerblockshelpprotecttheembryosasyougothruallthestepsandtheyarealsomucheasiertosee(andthereforetoavoidsuckingintoyourpipetteanddiscarding).Thedistinctshapesallowyoutodehydratealltheembryosinthesamevialwithoutconfusingthem.

17.RinsetheblocksindH20andtransfertoavial.Iusedascintillationvial.

18.Dehydration:

6"30%EtOH

10"50%EtOH

10"70%EtOH

*..70%EtOH[ifyouneedtotakeabreak,thisisasafeplacetohaltprocessingovernight]

6"90%EtOH

6"95%EtOH

3X20"100%EtOH

20"1:1PropyleneOxide:EtOH

2X8"PropyleneOxide

30"PropyleneOxide:Araldite2:1

60"PropyleneOxide:Araldite1:2

2X30"AralditeRoomTemp

Trimtheblocksandreturnembryostoseparatewellsofa9wellplate.

24hrsAralditeRT

Transferblockstowhatevermoldyouwillusetoholdtheembryoandtryyourbesttoorientembryoforeasiesttrimmingandcutting

3daysAraldite@60degreesC

BufferRecipes

0.2MNaCacodylate

A42.8gNa(CH3)2As023H2O

1000mldH2O

B0.2MHCl[10mlconcHCl+603mldH2O]

50mlA+B/18.3->pH/6.4Q.S.to200ml

Pretesttheosmolarity

CarolynNorris"recentupdatetothismethod[duringtheJune4workshopinMADIson]stressestheimportanceofosmolaritytrialstodiscoverthecorrectamountofsucroseinthefixativetomatchtheembryo"sinherentosmolarity:

Beforefixingthefinalspecimens,sherecommendsdoingseveraltrialembryoswithdifferingamountsofsucroseinthefinalfixative.Whilewatchingunderthemicroscope,sheshootsseverallaserholesintheeggshell.Iftheembryocollapsesrapidly(implodes),thenthesucroseconcentrationistoohigh.Iftheembryoblowsuprapidly(explodes),thenthesucroseistoolow.Underoptimalconditions,theembryowillbeseentoswellinvolumeveryslowlyafteralaserholeisputthroughtheeggshell.Thusthefinalfixativeshouldbetestedempiricallyonthedayoffixation,tooptimizetheexactosmolarity.