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		<h2>Fixations/Mounting</h2>
			<p>Fixation protocols may differ according to the treatments the embryos have been given or will be given next. 
			Regular fixation protocols</p>
			<ol>
				<li>Incubate 30 min. to overnight in 4% paraformaldehyde in a buffer, pH 7.4 (any 0.1M Tris, HEPES, PO4 buffers).</li>
				<li>rinse for 30 min in any of the buffer used </li>
				<li>Store in buffer at 4oC or mount in 70% glycerol </li>
			</ol>
			<p><h3>Zamboni's Fixative:</h3>
			<ul>
				<li>200ml of 8% parformaldehyde made up in dH20 </li>
				<li>100ml of 0.4M phosphate buffer </li>
				<li>30ml of saturated picric acid solution (1.5%) </li>
				<li>Add dH20 to make a total of 400ml </li>
			</ul>
			</p>
			<p><h3>Relaxing embryos before fixing</h3>
			This procedure aims at inhibiting muscular contractions that "freeze" the embryo at crooked position if transferred directly from Htr into fixing solution. Such crooked position may pose a serious problem when dissecting the main body part and trying to mount it flat between slide and coverslip.
			</p>
			<ul>
				<li>Embryos Injected With Dextran Cell Tracers:<br>
				Use "relaxant Htr medium" which contains 8% ethanol (which literally makes the embryos drunk) for 10-15 minutes prior to fixing.</li>
				<li>Embryos Injected With Di I:<br>
				DON'T use "relaxant Htr medium"!  It contains ethanol that leads the Di I to diffuse from a specific cell lineage into the surrounding medium and eventually disappears altogether from the cells.</li>
			</ul>
			<ol>
				<li>Relaxing with CO2: put a bloc of dry ice in an vacuum flask, seal the main opening and put a rubber pipe on the nosel. Adapt either a pipette tip or a pasteur pipette at the end of the tube and dive the tip into the Htr containing the embryos to relax wait until the embryos stop moving. Remove the Htr and replace with cold fixative solution (4oC) or transfer embryos to cold fixative solution.</li>
				<li>Relaxing at cold temperature: Put the petri dish or container with embryos on ice and check until embryos stop moving, then add cold fixative solution (4oC).</li>
			</ol>
				
		<h2>Wholemounting Embryos in BBBA</h2>
		<p><h3>Materials</h3>
		<ul>
			<li>BBBA*** = 3:2 mixture of benzyl benzoate: benzyl alcohol </li>
			<li>0.1 M Tris buffer pH 7.4 = Dilute from 1.0M stock (*see below) and adjust pH using 1 M NaOH or 1 M HCl as required.</li>
		</ul>
		* 1.0M Tris stock, pH 7.5: 240g Trizma-HCl and 47.2g Trizma-base made up to 2 liters  in ddH2O, autoclave.<br>
		*** BBBA is extremely corrosive! The only containers that will hold it are glass, or microfuge (Eppendorf) tubes. <br>
		<h2>Method </h2>
		<ol>
			<li>Fix embryos for 3h at room temperature or overnight at 4¯C in 3.7% formaldehyde in 0.1 M Tris buffer pH 7.4. 
		- Wash 3-4 times in Tris to remove formaldehyde.</li>
			<li>If Hoechst staining is desired, use 1:200 (5 ul/ml) dilution of Hoechst 33258 (1 mg/ml) in Tris. <br>
		Incubate for exactly 1.5 h in refrigerator.  Rinse in Tris.</li>
			<li>Dehydrate in an ethanol series: 30, 50, 80, 95, 100%, 1-2 minutes in each.</li>
			<li>Take embryos from 100% ethanol and drop into BBBA in a glass depression slide or dish.  Embryos will become transparent.</li>
			<li>Embryos can be stored in BBBA at 4¯C or -20¯C for months.</li>
			<li>Embryos are then mounted (temporarily) on a glass slide, and coverslip is held aloft by a thin silicon slat.</li>
		</ol>
		</p>