C. Collection of Embryos
1. Worms are grown on a lawn of E. coli strain OP50 on NGM agar plates (100 x 15 mm). For the wild type strain N2, each plate is started with 20 adult hermaphrodites which are allowed to grow until all their progeny have started to lay eggs. Ten such plates yield enough embryos for approximately thirty individual wells. Chunks of agar on plates should be avoided, since these can interfere with the freeze-cracking step.
2. Gravid hermaphrodites and their laid eggs are washed off the plates in water, and collected into a 15 ml conical tube. After centrifugation (1700 rpm for a sufficient time to pellet embryos and adults, approximately 1min), most of the water above the pellet is removed.
3. The worm pellet is resuspended in 10 ml of hypochlorite solution, and incubated at room temperature for 3 minutes with some agitation. Worms are recovered by centrifugation and incubated in fresh hypochlorite solution for another 3 minutes (Incubation times may vary depending on the type of bleach used). Embryos are protected by their egg shell from hypochlorite digestion, while larvae and adults are dissolved by this treatment.
4. When carcasses of larvae and adults are no longer visible in the dissecting microscope, embryos are washed twice in 15 ml of PBS, before resuspending in a small volume of PBS (0.5 ml or less).
D. Permeabilization and fixation of embryos
1. Embryos are transferred to the polylysine-coated slides using a micro-pipet. (15 µl of embryos in PBS is sufficient to cover a 14 x 14 mm square well). Make sure the embryos are not forming clumps. Clumps can be separated by blowing air onto the embryos through a micropipet.
2. Embryos are overlayed with a glass coverslip and the slide is placed in a humidity chamber. Repeat for all slides
Modification of protocol for larvae and adults:
Wash worms in PBS, spin them down, resuspend in small volume of PBS, and spread 5ul containing 10 to 50 worms onto the polylysined 14x14mm well. Cover the worms with a coverslip making sure that the PBS spreads onto the painted part of the slide thus pulling the coverslip down. You can then press on the coverslip ever so lightly to burst open the hermaphrodites, so as to obtain best permeabilization of the germline and embryos. Proceed with freeze-cracking (step 3).
3. Slides are frozen by placing on an aluminum block that has been pre-cooled on dry ice. The coverslips are then quickly snapped off, and the slides are immediately immersed in 100% methanol at -20oC for 5 min.
3. Slides are transferred to 100% methanol at room temperature for 5 min and then rehydrated at room temperature as follows :
- One 1 min wash in 90% MeOH in H2O.
- One 1 min wash in 70% MeOH in PBS.
- One 1 min wash in 50% MeOH in PBS.
- Two 5 min washes in PTw
IF YOU DO NOT PLAN TO DO THE PROTEINASE K DIGESTION STEP, YOU CAN SKIP THESE LAST TWO PTw WASHES AND GO DIRECTLY FROM 50% MeOH TO THE FIXATIVE SOLUTION (step5).
4. At this point, a Proteinase K digestion step can be incorporated. Such a step may be necessary to increase the accessibility of low abundance messages expressed after the lima-bean stage (Pete Okkema, pers. communication). However, for earlier stages, proteinase K treatment is not recommended. Each batch of proteinase K needs to be titrated to determine proper incubation conditions. (Typically, a 15 min incubation in a 1 µg/ml solution in PTw at room temperature is sufficient if needed). Proteinase K digestion is stopped by incubating for 2 min in 2mg/ml glycine in PTw, followed by two 5 min washes in PTw. (If protease digestion is not necessary, proceed directly from step 3 to step 5).
5. Embryos are fixed by incubation at room temperature for 20 min in formaldehyde fixative solution.
6. To remove formaldehyde, embryos are washed extensively at room temperature as follows:
- Two 5 min washes in PTw.
- One 5 min wash in 2 mg/ml glycine in PTw.
- Three 5 min washes in PTw.
E. Prehybridization
1. Fixed embryos are incubated for 10 min in a 1:1 mix of hybridization buffer and PTw, followed by a 10 min incubation in undiluted hybridization buffer. (These incubations are done at room temperature). During this time, a separate 150 ml aliquot of hybridization buffer is heated in a boiling water bath for 10 min, and cooled on ice.
2. Embryos are pre-treated in this freshly-heated hybridization buffer for 1-2 hours at the temperature to be used for probe hybridization (48oC for cDNA-derived probes and 37oC for oligonucleotide probes).
F. Hybridization
1. cDNA-derived probes can be used undiluted (original 300µl, appr. 5 µg/ml) or up to nine-fold diluted depending on the abundance of the transcript. In general, the probe is diluted 3-fold for moderately abundant messages (eg. skn-1 mRNA) and up to nine-fold for very abundant messages (eg. unc-54 mRNA; lacZ mRNA derived from a multiple-copy array). Oligonucleotide probes are used at a concentration of 0.5 µg/ml.
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