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