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		<h3>Antibody staining</h3>
			<ul>
			<li>Fix embryos with 4% formaldehyde in 0.25XPBS for 60 to 90 minutes (max) at room temperature. For embryos at mid stage 9 or older, incubate them in relaxant medium for 5 minutes at 4o C prior to fixing them with cold fixative (same fixative as above).</li> 
			<li>Rinse with PBT (1X PBS, 1% triton 100). </li>
			<li>Devitellinize embryos in PBT . (Can save the embryos in PBT for a week)</li> 
			<li>Treat embryos for 40 minutes with 50ul of protein kinase (1ul PK 20mg/ml in 1600ul 1XPBS) at room temperature. </li>
			<li>Pre-incubate embryos with 100ul of Antibody serum* (1XPBS, 10% NGS, 1% triton X-100) for several hours (3-6hrs) at room temperature or O/N at 4o C </li>
			<li>Replace with ~250-500ul of primary mouse anti-histone antibody (1ul of anti-histone antibody in 1ml antibody serum). Incubate overnight with constant rolling, at room temperature. </li>
			<li>Rinse every hour with 100ul antibody serum for 4 to 8 hours. </li>
			<li>Replace with secondary HRP conjugated rabbit anti-mouse antibody (1ul of secondary antibody in 500ul antibody serum). Incubate O/N at room temperature with constant rotating, same as for primary antibody). </li>
			<li>Rinse once every hour for 2 hours at room temperature. </li>
			<li>Do HRP reaction immediately after rinsing.</li>
		</ul>
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		<h3>Horse Radish Peroxidase (HRP) staining</h3>
		<ul>
			<li>Wear gloves </li>
			<li>Get an aliquot of DAB in the -20 freezer. </li>
			<li>Each aliquot contains 250ul of 2mg/ml DAB. Centrifuge the aliquot so that the DAB solution is located at the bottom of the tube. Add 750ul of PBS. </li>
			<li>Make a solution of 1%-3% H2O2 in an eppendorf tube. </li>
			<li>Prepare waste container with bleach in it. </li>
			<li>Take a 12 wells porcelain dish and put 100ul of DAB in each well </li>
			<li>Transfer embryos to the wells, incubate 10-20 minutes in DAB. Cover wells with aluminum foil when incubating. </li>
			<li>Add 3ul of 1% H2O2 in each well and monitor the embryos in each well for staining. </li>
			<li>When desired staining coloration is reached, pipette out the DAB (make sure to leave the embryos in the well) and discard DAB into the waste bottle containing bleach. </li>
			<li>Rinse the embryos with PBS. </li>
			<li>Transfer embryos in eppendorf tube with fresh PBS. </li>
			<li>Embryos are ready for dehydration or for transfer in glycerol. </li>
		</ul>

		 
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