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		<h3>Silver Staining </h3>
		<p>(As amended by Francoise Z. Huang)
		
		
		<h2>Materials</h2>
		<ul>
			<li>2% formaldehyde in 0.1 M tris-HCl (pH 7.5) </li>
			<li>ddH2O </li>
			<li>silver methenamine solution [0.1% AgNO3, 1% hexamethylene tetramine, and 0.25 M boric acid (pH 9.4)]. Must be freshly made. </li>
			<li>1 or 2 fiber optic lamps </li>
			<li>4% formaldehyde in 0.1 M tris-HCl (pH 7.5)</li>
		</ul>
		<h2>Method </h2>
		<ol>
			<li>Embryos are fixed in 2% formaldehyde in 0.1 M tris-HCl (pH 7.4) for 5 minutes at room temperature. </li>
			<li>Rince embryos briefly in distilled water to remove excess ions </li>
			<li>Transfer embryos in in silver methenamine solution [0.1% AgNO3, 1% hexamethylene tetramine, and 0.25 M boric acid (pH 9.4)]. </li>
			<li>Directly expose embryos to strong, white light from 1 fiber optic lamps (Dolan Jenner Industries) until the superficial cells were outlined by deposition of a dark brown product along the furrows of the cells (about 5-10 minutes). </li>
			<li>Rince embryos briefly in distilled water to remove excess silver </li>
			<li>Embryos are then fixed for 1h in 4% formaldehyde in 0.1 M tris-HCl for 1h at room temperature. </li>
			<li>Rince briefly with 0.1 M tris-HCl. (Embryos can also be rinced with 0.1 M tris-HCl containing Hoechst for 15 minutes).</li> 
			<li>Remove rinse and put 40% glycerol (leave it there until embryos start to sink to the bottom of tube). Then transfer them to 100% glycerol. </li>
			<li>Embryos are ready for viewing or to be stored (4o C or -20o C).</li>
		</ol>
			NB: For devitellinized embryos beginning silverstaining procedure at step 2, the Htr medium must be rinsed from the embryos entirely before incubation in the silver solution to prevent the precipitation of silver chloride 
		</p>