Transplantation of the eyeforming region of Amphibian neurula into the flank of an albino embryo相关交流-蚂蚁淘在线

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EMBRYOCELLANDTISSUECULTURETECHNIQUES

Rearingsolution:10%ModifiedSteinberg"sSolution.Operatingsolution:FullstrengthHBStsolution.

MicrosurgeryPreparation

1)Placetwoembryosinthe60mmpetridishcontainingOperatingsolutionwithantibiotics.2)Usingtweezersandforcepsdejellytheembryoandremovethemembranearoundit.3)Prepare2%agarose-coatedoperatingdishesin1XHBSt(+/-powderedcharcoalforcontrast),rinsewithHBStthatcontainsantibiotics.4)Placeembryosintooperatingdishcontaining1XHBStandantibiotics.Usehairloopstotransfertheembryos.Thedonorandrecipientembryosshouldbeoperatedonseparatedishes.Placethedonoronapetridishwithclearagarosegel,andthealbinorecipientonanagarosepowderedwithcharcoal.

RemovaloftheEyeFormingregion:ModifiedfromHamburger,V.1960.AmanualofExperimentalBIOLOGy.TheUniversityofChicagoPress,pp.107-109.1)PositionthedonorembryosothattheanteriorpartoftheneuralfoldiscrearlyvisIBLe.2)Usingeye-browknivesmakeveryshallowcutsintheanteriorportionoftheembryo.3)Afterthecutsaremadecarefully,nottotairtheentireembryoapart,takeoffaverythinlayerofthetissue.

PreparationofAlbinoRecipientEmbryo:

1)Placethealbinoembryoswiththeflankafreafacingup.2)Makeanincisionstartingfromtheanteriortotheposterioroftheembryo.3)Carefullytransferthegraftstothepetridishcontainingthealbinoembryo.4)Placethegraftintotheflankcutofthealbino.Makesureitstaysinplacesothatthewoundcanheal.5)Observetheembryosafterafewdaystoseewhetherthegraftshavedevelopedintoaneye.Takedigitalimagesofthegraftedembryos.

^{Usecleantechnique;diptoolsandPipettesin70%EtOHandthenintosterileHBStbeforeusing.}

Results:

Thetransplantadhered,andtheeyeformationtookplace.Itwasnecessarytomakesurethatthetransplantadherestotheflankofthealbino,otherwisethewoundwillnotheal,andthetransplantwilldissociatefromtheembryo.Duringtheexperimenttheembryocontinueditsdevelopmentuptothestage36.Sinceatthestage16thecellsintheeyeformingregionarealreadycommittedtobecomeneuralcells,thetransplantedpatchwasabletoformindependently,regardlessofitsnewposition.

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