EdU labeling and fixation
(Modified by Schedl lab from protocol provided by Sarah Crittenden)
To label on plates: (from Ito and McGhee, 1987)
1. Grow an overnight of MG1693 (thy-, from E. coli stock center)
2. To 100 ml of M9, add:
~2 ml ON MG1693
5 ml 20% glucose (0.4%-1% – usually 1%)
100 ul 1.25 mg/ml thiamine (1.25 ug/ml)
100 ul 0.5 mM thymidine (0.5 uM)
100 ul 1M MgSO4 (1mM)
200 ul 10 mM EdU (Invitrogen) (20 uM)
Grow at least ~24 hours at 37˚C. Spin to pellet E. coli. Resuspend in about 1 ml M9.
3. Seed plates with labeled MG1693 on M9 agar plates. They take about a day to dry.
4. Put worms on plates.
5. Wash worms off with PBS (it is difficult to pick worms off the filamentous MG1693) and process.
For co-staining with DAPI or antibodies:
- Dissect and fix for <5 minutes in 3% paraformaldehyde/ 0.1 M K2HPO4 (pH 7.2), 5 min. Post-fix in ice cold MeOH [although EdU is compatible with basically any fix, we’ve found that this works best].
- wash 3X with PBT
- move to antibody staining (if you’re going to stain)
- DAPI stain
- As a last step, do the Click-IT EdU reaction according to the instructions provided by Invitrogen, except do 2×30 minute incubations. Doing the EdU reaction before antibody staining can interfere with staining for some antibodies.
- Wash 3X in PBS or PBT and mount.