FreezeFracture for Immunofluorescence of Embryos
来自 : 蚂蚁淘
Materials:disposablescapel | #15(roundtip)featherbrand |
| |
subbedslides | 0.1%poly-L-lysine(MW>300,000) |
| 0.2%gelatin |
| 0.01%chromealum |
| (dipslidesinsolutionanddryovernight) |
| |
Whatmannpaper | foreasecutinto1inchsquarepieces |
| Procedure:- Dissectembryosfromgravidhermaphrodites
- pickgravidhermaphroditesinto100ulofH20onasubbedslide
- cuthermaphroditesnearvulvawithdisposablescapeltoreleaseembryos
- Freeze-fractureembryos
- removepracticallyallofliquidfromslidewithpipet(~90ul)
- placecoversliponembryos/wormcarcasses
- wickresidualliquidoutwithWhatmannpaperuntilembryosbegintoflatten
- freezeslideondryice>20minutes
- quicklyflipoffcoverslipwithrazorblade
- immerseslideinfixative
- processforimmunofluorescence
Notes:- Mostproblemsareduetolackofpermeablizationofembryoswhennotenoughpressureisapplied
- Useyoungeradultstoenrichforearlierembryos
- Multiplecutswiththescapelcanextrudeearlierembryosorembryosstillcaughtintheuterus(aftertheinitialcut,beginneartipand"push"embryosoutliketoothpastefromatube)
- Forosmoticallysensitiveembryos,dissectin150mMsalt
- Ifthescapel"pulls"toomuchliquidwhencutting,drythescapelonaKim-wipe
- Topreventthecoverslipfromfallingbackontoembryos,flickthecoverslipoffwhileholdingtheslidewiththecoverslipsidedown
- reference:TheNematodeC.elegans(Wood,1988):Methods(p.587-606)
compiledbyChadRappleye AroianLabProtocols |
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