Schedl Lab

Washington University Genetics

EdU labeling and staining

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:

  1. 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].
  2. wash 3X with PBT
  3. move to antibody staining (if you’re going to stain)
  4. DAPI stain
  5. 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.
  6. Wash 3X in PBS or PBT and mount.
  • Tim Schedl, Ph.D
    Department of Genetics
    Campus Box #8232
    Washington University School of Medicine
    4566 Scott Ave.
    St. Louis, MO 63110

    Contact

    Email: ts@genetics.wustl.edu
    PHONE: (314)362-6164 [lab]
    FAX: (314)362-7855