BUdR visualization:
1) Dissect cephalic complexes in PBS and fix for 30 minutes at room temperature in
Carnoy's fix; (60 ml ethanol + 10 ml glacial acetic acid + 30 ml chloroform).
2) Rehydrate by incubating for 3 minutes each in 70%, 50%, 30% ethanol in PBS-TX (1xPBS, 0.3% Triton X-100). Finish by incubation in PBS-TX for 5 minutes on ice.
3) Denature the DNA by incubating for 1 hr in 2N HCl/PBS-TX (1:1 mix of 4N HCl and PBS-TX). Wash twice, 5 minutes each, in PBS-TX on ice.
4) Incubate for 30 minutes in 10% goat serum in PBS-TX.
5) Incubate overnight at 4°C (I imagine that for eye-discs, in contrast to whole embryos or larval CNSs, a few hours is sufficient) in the primary antibody (1:200 dilution of monoclonal anti-BUdR from Becton-Dickinson).
6) Wash in PBS-TX on ice six times for 10 minutes each (again, this might be an exageration when staining eye discs).
7) Incubate at 4°C for several hours to overnight in a 1:200 dilution of biotinylated goat anti-mouse IgG (Vectastain;) in PBS-TX.
8) Wash in PBS-TX on ice six times for 10 minutes each.
9) Incubate for 30 minutes in Vectastain ABC reagent (10µl of reagent A + 10µl of
reagent B + 980µl of PBS).
10) Wash in PBS on ice two times for 10 minutes each,
11) React with DAB/H2O2/CoCl2 (100µl 5mg/ml DAB + 900µl PBS + 1µl 3% H2O2 +
20µl 1% CoCl2) until stain becomes evident, usually a few minutes.
12) Dehydrate discs by a graded series of 30, 50, 70, 90, 100% ethanol.
13) Mount in DPX.