<p class="jove_content">We thank Drs. Richard H. Masland and Akimichi Kaneko for giving us valuable advices and suggestions to establish the procedure of the horizontal slice preparation of the retina.</p>

<ol>
<li>Prepare an agar block (30 mg/ml, ie 3%, in distilled water). Dissolve and microwave it first and pour it into a glass dish. Keep the dish in a refrigerator at 4 <sup>o</sup>C.<br /><em>Tip: The agar block should be prepared in advance.<br /></em><br /></li>
<li>Prepare low-temperature-melting agarose gel (25 mg/ml, ie 2.5%, in medium). Dissolve and microwave it. Keep it in the hot water bath at 35 <sup>o</sup>C.<br /><em>Tip: Keep the agarose gel NOT too hot. If it is too hot, it takes a long time to be solidified, and the gel may kill...

<ol>
	<li>Azuma, T., Enoki, R., Iwamuro, K., Kaneko, A., Koizumi, A., Koizumi, A., TC, J. a. k. o. b. s., Masland, R. H., MA, M. a. c. N. e. i. l., RH, M. a. s. l. a. n. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+spatiotemporal+patterns+of+dendritic+Ca2++signals+in+goldfish+retinal+amacrine+cells.">Multiple spatiotemporal patterns of dendritic Ca2+ signals in goldfish retinal amacrine cells.</a> <em style='font-style: italic'>Brain Res</em>. , 64-73 (2004).</li></ol>

<p class="jove_content">All experimental protocols were conducted according to the National Institutes of Health guidelines for animal use.</p>

<p class="jove_content">There are several important advantages of the horizontal slice preparation of the retina.</p>
<p class="jove_content">First, the morphology of the cells that expand dendrites horizontally, such as amacrine cells and horizontal cells, is preserved. It has been shown that there are over 20 types of amacrine cells in the retina (MacNeil and Masland, 1998), so that it is quite important to compare the morphology of the cells with the physiological property.</p>
<p class="jove_content"> Second, in the whole-mount retina, th...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-1296</td>
<td>&nbsp;</td>
<tr>
<td>Agarose L</td>
<td>&nbsp;</td>
<td>Wako</td>
<td>317-01182</td>
<td>Low-temperature melting agarose </td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>Type I-S, 300 units/mg</td>
</tr>
<tr>
<td>Medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>in mM: 102 NaCl, 3.1 KCl, 2.0 CaCl2, 1.0 MgCl2, 23 NaHCO3, and 10 glucose, maintained at pH 7.8, or, Ames&#x2019; Solution (Sigma)</td>
</tr>
<tr>
<td>Microwave</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hot water bath</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>35 C</td>
</tr>
<tr>
<td>Vibratome slicer</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>VT1000S</td>
<td>&nbsp;</td>
<tr>
<td>MF-Membrane filters</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>HAWP01300</td>
<td>Filter paper , pore size 0.45 um</td>
</tr>
<tr>
<td>Dissection tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>forceps, blade, pinch, twizzer, etc.</td>
</tr>
<tr>
<td>Instant glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>eg., Alonalpha, Krazy Glu</td>
</tr>
<tr>
<td>50 ml syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>syringe filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SX0001300</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

horizontal slice preparation of the retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Watch this Scientific Journal Video about Horizontal Slice Preparation of the Retina at JoVE.com

Horizontal Slice Preparation of the Retina

<p class="jove_content">Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.</p>

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

horizontal-slice-preparation-of-the-retina

Research

JoVE Journal

Biology

15.6K Görüntüleme.  Dalhousie University. Geleneksel dikey dilim ve retinanın tüm hazırlık retina devrelerin fonksiyonunu incelemek için kullanılmıştır. Burada, sağlam retina nöronların dendritik morfoloji korumak için yöntem dilimleme roman açıklar.