TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved <em>Drosophila melanogaster</em> transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.</p>

[BEFORE_START]
<p><strong>Reagents and Supplies</strong></p>
<p><strong> </strong></p>
<ul>
<li>AF594 Donkey α- Jackson Immuno Research.  # 711-585-152  </li>
<li>ATTO 647N Goat α-Rat IgG (H&amp;L) Antibody. Rockland. # 612-156-120</li>
<li>DL550 Mouse α-V5 Tag. AbD Serotec.  # MCA1360D550GA</li>
<li>DPX Mountant for Microscopy. Electron Microscopy Sciences. # 13512, 500 mL</li>
<li>Ethanol, ACS reagent, &gt;99.5% (200 proof).  Sigma Aldrich. # 459844-1L</li>
<li>GS – Goat Serum. Life Technologies. 16210-064, 100 mL</li>
<li>Kodak Photo-Flo 200 Solution. Electron Microscopy Sciences. # 74257</li>
<li>Cy2 Snap ligand, Luke Lavis, JRC</li>
<li>NMS – Normal Mouse Serum. Jackson Immuno Research. # 015-000-120</li>
<li>PBS – Phosphate Buffered Saline, 1X<strong>. </strong> # 21-040</li>
<li>PFA – Paraformaldehyde. 20% PFA.  Electron Microscopy Sciences.  # 15713-S</li>
<li>Poly-L-Lysine. Sigma Aldrich. # P1524-25MG</li>
<li>Protein LoBind Microcentrifuge Tubes - 2 mL. # 022431102</li>
<li>S2 – Schneider’s Insect Medium. Sigma Aldrich. # S01416</li>
<li>Rabbit α-HA Tag. Cell Signal Technologies. # 3724S</li>
<li>Rat α-FLAG Tag (DYKDDDDK Epitope Tag). Novus Biologicals.  # NBP1-06712</li>
<li>Triton X-100. Sigma Aldrich. # X100</li>
<li>Fisher Scientific. # X5-500</li>
</ul>

[GUIDELINES]
<ul>
<li>All tissues and solutions are at room temperature (RT), unless noted. Always protect tissue from light exposure.</li>
<li>For details on dissection and fixation see FlyLight Protocol - Adult Dissection and 2% Fixation.</li>
<li>For mounting and embedding instructions refer to FlyLight Protocol – DPX Mounting.</li>
<li>Protocols at <a href="https://www.janelia.org/project-team/flylight/protocols" target="_blank" rel="noopener noreferrer">https://www.janelia.org/project-team/flylight/protocols</a></li>
</ul>
<ul>
<li>
<p><strong>Reporter Genotype: </strong>pBPhsFlp2::PEST in attP3; brp-SNAP / CyO; pJFRC201-10XUAS-FRT&gt;STOP&gt;FRT-myr::smGFP-HA in VK0005, pJFRC240-10XUAS-FRT&gt;STOP&gt;FRT-myr::smGFP-V5-THS-10XUAS-FRT&gt;STOP&gt;FRT-myr::smGFP-FLAG in su(Hw)attP1</p>
</li>
<li>
<p>For details on reporter constructs see Nern, et al., 2015.</p>
<p><a href="http://www.pnas.org/content/112/22/E2967.long" target="_blank" rel="noopener noreferrer">http://www.pnas.org/content/112/22/E2967.long</a> doi: 10.1073/pnas.1506763112</p>
</li>
<li>
<p>For details on brp-SNAP chemical tagging, please refer to Kohl, et al., 2014. <a href="http://www.pnas.org/content/111/36/E3805.long" target="_blank" rel="noopener noreferrer">http://www.pnas.org/content/111/36/E3805.long</a> doi: 10.1073/pnas.1411087111</p>
</li>
</ul>

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