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			<h2>Br-dUTP  Incorporation and Anti Br-dUTP Immunochemistry</h2>
			(S.T. BISSEN, Development 105: 105-118)
			(Adapted to H. robusta, D.A. LANS)

			<h2>Materials</h2>
			<p>
			<ul>
				<li>5-bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP; Sigma, 50mM in 0.2 M KCl, 0.5% fast green FCF)</li>
				<li>2% formaldehyde (in 50 mM cacodylate buffer, pH=7.3)</li>
				<li>50 mM cacodylate buffer, pH=7</li>
				<li>phosphate buffer saline (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH=7)</li>
				<li>2N HCl in PBS</li>
				<li>0.1 M sodium borate, pH=8.5</li>
				<li>TBP (1% Triton X-100 and 2% bovine serum albumin in PBS)</li>
				<li>antibodies:</li>
				<li>mouse monoclonal anti BrdU antibody (1:25 in TBP)</li>
					<ol>
						<li>a) Rhodamine/fluorescein-conjugated rabbit anti-mouse IgG (ICN, 1:500 in TBP)</li>
						<li>b) or HRP-conjugated goat anti-mouse IgG (Polysciences, Inc., diluted 1:50 in TBP). in case 2), you will also need:</li>
							<ul>
							<li>Peroxydase-anti-peroxydase complex (PAP, Polysciences, Inc.)</li>
							<li>H2O2</li>
							<li>Diaminobenzydine (DAB), 0.5mg/ml)</li>
							<li>0.1 M Tris pH=9</li>
							<li>Hoechst 33258</li>
							<li>80% Glycerol (20% 0.1 M Tris pH=9)</li>
							</ul>
					</ol>
			</ul>

			<h2>BrdU incorporation and fixation</h2>
			<ul>
			<li>Pressure inject</li>
			<li>BrdUTP and incubate embryos for the delay decided.</li>
			<li>Fix in 2% formaldehyde in cacodylate buffer pH=7.3 for 1h in 4o C</li>
			<li>Rinse in 50 mM cacodylate buffer</li>
			<li>Transfer into PBS</li>
			<li>Manually devitellinize embryos</li>
			<li>Incubate in 2N HCl-PBS for 1.5h at room temperature (partially denature the DNA)</li>
			<li>Rinse briefly in 0.1 sodium borate, pH=8.5 (neutralise acid)</li>
			<li>Rinse in PBS</li>
			<li>Incubate in TBP overnight (or 6-8h) at room temperature</li>
			<li>Embryos are now ready for staining</li>
			</ul>

			<h2>Staining</h2>
			<p>Every incubation and rinsing are proceeded with gentle rocking
			<ul>
			<li>Incubate in mouse monoclonal antibody-TBP (1:25) overnight (16h) at room termperature</li>
			<li>rinse in TBP for 6-8hrs</li>
				<ol>
				<li>Incubate in ICN-TBP (1:500) overnight (16h) at room To on a platform shaker.<br>
			- Rinse in PBS for 6-8hrs<br>
			- Stain for 1h with 1mg/ml Hoechst 33258 in H2O<br>
			(alternatively, by D. Lans:<br>
			- Wash in 0.1 M Tris(pH=9)<br>
			- Stain for 1h with 1mg/ml Hoechst 33258 in Tris-HCl, pH=9)<br>
			</ol>
			<ol>
			<li value= "2">Or incubate in goat anti mouse IgG-TBP (1:50) overnight (16h)<br>
			- rinse in TBP for 6-8h<br>
			- Incubate with 50 mg/ml peroxidase-anti-peroxidase complex<br>
			- rinse in PBS<br>
			- Proceed with H2O2 in presence of 0.5mg/ml DAB to yield dark reaction product<br>
			- mount<br></li>
			</ol>
			</ul>
			<h2>Mounting</h2>
			<ul>
				<li>Mount  in 80% glycerol (80% glycerol + 20% Tris buffer) or</li>
				<li>Plastic-embed embryos and section</li>
			</ul>
			Before observation of any fluorescent dyed embryos, they can be treated with n-propyl gallate, it slows the bleaching and hence increases the delay of observing time. Those embryos can also be mounted in 80% (and above) glycerol, the pH is more basic which slows the bleaching.
			
	
		</p>