BrdU Labeling of Larval Eye Discs

  1. Dissect larval eye discs in Schneider’s medium (RT room temperature).
  2. Transfer into Schneider’s medium containing 0.1 ~ 0.25 mg/ml BrdU at 25°C.
  3. Incubate 30 minutes for first and second mitotic wave or 90 minutes to
 capture ectopic S phases posterior to the morphogenetic furrow.
  4. Wash once in RT Schneider’s medium.
  5. Wash twice in PBS on ice.
  6. Fix over night at 4°C in 1% paraformaldehyde + 0.01 % tween-20.
  7. Wash 5 times with PBS.
  8. DNase treat: Incubate 5ul RQ1-DNAse (1ul/ul)(Promega) + 95 ul DNAse
 Buffer for 2 hours at 37°C (water bath).
  9. Wash twice with PBT.
  10. Incubate 1 hour with mouse anti-BrdU (1:50) in PBT.
  11. Wash 3 times in PBT.
  12. Incubate 1 hour at RT with anti-mouse secondary antibody (or overnight at 4°C).
  13. Wash in PBT 3times fast. Then 4 times 20 minutes each.
  14. Mount discs.

    Fixation solution: 1x PBS, 1% paraformaldehyde, 0.01% tween-20.
DNase buffer : 40 mM Tris (pH 8.0), 10 mM MgSO4, 1mM CaCl2.
PBT: 1x PBS + 0.3% triton X-100