Br-dUTP Incorporation and Anti Br-dUTP Immunochemistry
(S.T. BISSEN, Development 105: 105-118) (Adapted to H. robusta, D.A. LANS)
Materials
- 5-bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP; Sigma, 50mM in 0.2 M KCl, 0.5% fast green FCF)
- 2% formaldehyde (in 50 mM cacodylate buffer, pH=7.3)
- 50 mM cacodylate buffer, pH=7
- phosphate buffer saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH=7)
- 2N HCl in PBS
- 0.1 M sodium borate, pH=8.5
- TBP (1% Triton X-100 and 2% bovine serum albumin in PBS)
- antibodies:
- mouse monoclonal anti BrdU antibody (1:25 in TBP)
- a) Rhodamine/fluorescein-conjugated rabbit anti-mouse IgG (ICN, 1:500 in TBP)
- b) or HRP-conjugated goat anti-mouse IgG (Polysciences, Inc., diluted 1:50 in TBP). in case 2), you will also need:
- Peroxydase-anti-peroxydase complex (PAP, Polysciences, Inc.)
- H2O2
- Diaminobenzydine (DAB), 0.5mg/ml)
- 0.1 M Tris pH=9
- Hoechst 33258
- 80% Glycerol (20% 0.1 M Tris pH=9)
BrdU Incorporation and Fixation
- Pressure inject
- BrdUTP and incubate embryos for the delay decided.
- Fix in 2% formaldehyde in cacodylate buffer pH=7.3 for 1h in 4°C
- Rinse in 50 mM cacodylate buffer
- Transfer into PBS
- Manually devitellinize embryos
- Incubate in 2N HCl-PBS for 1.5h at room temperature (partially denature the DNA)
- Rinse briefly in 0.1 sodium borate, pH=8.5 (neutralise acid)
- Rinse in PBS
- Incubate in TBP overnight (or 6-8h) at room temperature
- Embryos are now ready for staining
Staining
Every incubation and rinsing are proceeded with gentle rocking
- Incubate in mouse monoclonal antibody-TBP (1:25) overnight (16h) at room termperature
- rinse in TBP for 6-8hrs
- Incubate in ICN-TBP (1:500) overnight (16h) at room To on a platform shaker.
- Rinse in PBS for 6-8hrs
- Stain for 1h with 1mg/ml Hoechst 33258 in H2O
(alternatively, by D. Lans:
- Wash in 0.1 M Tris(pH=9)
- Stain for 1h with 1mg/ml Hoechst 33258 in Tris-HCl, pH=9) - Or incubate in goat anti mouse IgG-TBP (1:50) overnight (16h)
- rinse in TBP for 6-8h
- Incubate with 50 mg/ml peroxidase-anti-peroxidase complex
- rinse in PBS
- Proceed with H2O2 in presence of 0.5mg/ml DAB to yield dark reaction product
- mount
Mounting
- Mount in 80% glycerol (80% glycerol + 20% Tris buffer) or
- Plastic-embed embryos and section