LineagingofC.elegansEmbryos

byPressure-freeMounting

---

Materials:

GlassSlides

18mmx18mmCoverslips

PetroleumJelly

#15DisposableScalpel

EmbryonicBlastomereMedium

Procedure:

1. Preparecoverslipswithpetroleumjellyspacer
   • applythinamountofpetroleumjellytothetopsurfaceofcoverslipalongallfouredges
2. Dissectembryosfromgravidhermaphrodites
   • pick3-5gravidhermaphroditestoaNGplatewithoutE.coli
   • placecoverslip("jellyside"up)ontopofglassslide
   • place8ulofmediumontopofcoverslip
   • using32gaugeplatinumpick,transferthehermaphroditesfromtheunseededplatetothedropofmedium
   • cuthermaphroditeswithscapeltoreleaseembryos
   • cutonbothsidesofthevulvatoreleaseearlierembryosandpushanyremainingonesoutofnematodecarcassbysuccessivepressuremovinginfromthetip
3. Prepareslide
   • gentlyplacesecondslideontopofcoverslip,allowingthemediumtospread
   • applyslightpressuretoedgesto"seal"thecoversliptotheslidewiththepetroleumjelly
4. ViewembryosbyDICmicroscopy

Hints&Miscellaneous

• Petroleumjellycreatessealtopreventdessicationaswellasspacingthecoverslipfromtheslidetopreventpressureonembryos
• Foreasyapplicationofpetroleumjellytocoverslip,prepare1mlsyringefilledwithmoltenjellyandpushthrough25gaugehypodermicneedle
• Canalsousetwo25gaugeneedlesin"scissor-like"motiontodissectembryosfromgravidhermaphrodites
• Fornon-pressure-freemounts,omitpetroleumjellyoncoverslipanduseglassslidewiththinpadof2%agaroseontopofcoverslip(alsoreducemediumto3ul)
• Foregg-shellpermeABIlitytests,includeNileBlueinmediumat5-10ug/ml

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