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.