TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Paraffin embedding and sectioning is a classic technique used in a variety of biological disciplines. Though the fruit fly </span><em><span style="font-weight: 400;">Drosophila melanogaster</span></em><span style="font-weight: 400;"> are one of the most commonly-employed model organisms, the immense </span><em><span style="font-weight: 400;">Drosophila </span></em><span style="font-weight: 400;">literature is lacking in current protocols for sectioning whole, adult flies. Here, we adopted a plant histology protocol to embed entire adult flies for thin sectioning and present our protocol for performing FISH on the subsequent sections. In the interest of reproducibility, we have tried to include details that are normally omitted from published protocols. </span></p>

[GUIDELINES]
<p>You will need the following reagents, consumables and equipment for each of the three steps as follows:</p>
<p> </p>
<p>I. Paraffin Infiltration of Adult Flies</p>
<p> </p>
<table>
<tbody>
<tr>
<td>
<p><strong>Reagents</strong></p>
</td>
<td>
<p><strong>Consumables</strong></p>
</td>
<td>
<p><strong>Equipment</strong></p>
</td>
</tr>
<tr>
<td>
<ul>
<li>Ethanol (VWR)</li>
<li>Chloroform (Fisher)</li>
<li>Acetic acid (Sigma)</li>
<li>ddH2O</li>
<li>Histoclear (National Diagnostics #HS2001GLL)</li>
<li>Paraplast paraffin (McCormick #39501006)</li>
</ul>
</td>
<td>
<ul>
<li>Glass scintillation vials (#FS74500-20)</li>
<li>Glass serological pipettes</li>
<li>Glass pasteur pipettes with rubber bulbs</li>
<li>Graduated plastic transfer pipettes (Fisher Scientific #13-711-9BM)</li>
<li>Embedding rings (Electron Microscopy Sciences #62350W)</li>
<li>Embedding base molds (Fisher Scientific #22-363-553)</li>
<li></li>
</ul>
</td>
<td>
<ul>
<li>Cold room or refrigerator</li>
<li>Orbital shaker</li>
<li>42C incubator</li>
<li>60C incubator or paraffin oven, shaking optional</li>
<li>Pyrex liquid measuring cups</li>
<li>Fine forceps (Dumont #5 or #55)</li>
<li>Teasing needle (or similar tool)</li>
<li>Pencil, for labeling</li>
<li>Microslide warming table (Eberbach corporation)</li>
</ul>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>II. Sectioning embedded flies via microtome</p>
<p> </p>
<table style="width: 659px;">
<tbody>
<tr>
<td style="width: 186px;">
<p><strong>Reagents</strong></p>
</td>
<td style="width: 254px;">
<p><strong>Consumables</strong></p>
</td>
<td style="width: 199px;">
<p><strong>Equipment</strong></p>
</td>
</tr>
<tr>
<td style="width: 186px;">
<ul>
<li>ddH2O</li>
</ul>
</td>
<td style="width: 254px;">
<ul>
<li>Graduated plastic transfer pipettes (Fisher Scientific #13-711-9BM)</li>
<li>818 high profile microtome blades (Leica Biosystems)</li>
<li>Razor blades</li>
<li>Polysine slides (Fisher Scientific #12-545-78)</li>
</ul>
</td>
<td style="width: 199px;">
<ul>
<li>42C incubator</li>
<li>Fine forceps (Dumont #5 or #55)</li>
<li>Teasing needle (or similar tool)</li>
<li>Pencil, for labeling</li>
<li>Microtome</li>
<li>42C heat block</li>
<li>Paraffin candle and lighter</li>
</ul>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>III. FISH on paraffin sectionsCold room or refrigerator</p>
<p> </p>
<table>
<tbody>
<tr>
<td>
<p><strong>Reagents</strong></p>
</td>
<td>
<p><strong>Consumables</strong></p>
</td>
<td>
<p><strong>Equipment</strong></p>
</td>
</tr>
<tr>
<td>
<ul>
<li>Ethanol (VWR)</li>
<li>Histoclear (National Diagnostics #HS2001GLL)</li>
<li>ddH2O</li>
<li>ProLong Gold with DAPI (Fisher Scientific #P36931)</li>
<li>Hydrochloric acid</li>
<li>Hybridization buffer (See Recipes)</li>
<li>PBSTx (See Recipes)</li>
<li>Fluorescently-labeled FISH probe*</li>
</ul>
</td>
<td>
<ul>
<li>60 x 24 mm Hybrislips ( Sigma-Aldrich #H0784)</li>
<li>60 x 24 mm #1 coverslips</li>
</ul>
</td>
<td>
<ul>
<li>37C incubator</li>
<li>Humid chamber#</li>
<li>Coplin jars or staining dishes</li>
<li>6” forceps</li>
<li>Fine forceps (Dumont #5 or #55)</li>
</ul>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p>*We’ve successfully used an 18mer DNA probe tagged with 5’ Alexfluor-647 (Thermo Fisher) that targets a ~11,000-fold copied sequence in genome.</p>
<p> </p>
<p><span style="font-weight: 400;">#Consists of wet paper towels and something to prop up slides above towels in a sealable plastic container. Construction described in protocol.</span></p>

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