TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The preparation and staining of samples for MERFISH follows closely the typical protocols used for smFISH (Raj et al., 2008). However, there are a few places in which we have modified these protocols to optimize MERFISH staining. Again, RNase contamination can destroy samples, so care should be taken to work in an RNase-free environment.</p>

[GUIDELINES]
<p style="text-align: justify;"><strong>The protocol workflow is as follows: </strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>1. Fixation and Permeabilization of Cells (Steps 1-5)</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">The first step in most FISH protocols is to fix the cells and then to permeabilize the membrane using either an overnight incubation in 70% ethanol or a brief exposure to surfactant. We prefer the latter approach since this protocol requires less time; however, we have had reasonable performance with both approaches.  </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>2. Hybridization of Encoding Probes (Steps 5-23) </strong></p>
<p style="text-align: justify;"> </p>
<p> </p>
<p><strong>This protocol will require the following reagents and buffers:</strong></p>
<p><strong> </strong></p>
<p>1. Fixation buffer (4% PFA in 1XPBS)</p>
<ul>
<li>1 mL 32% paraformaldehyde (PFA; Electron MicroscopySciences; 15714)</li>
<li>0.8 mL 10X Phosphate Buffered Saline (PBS; Ambion; AM9625)</li>
<li>6.2 mL nuclease-free water</li>
<li>Store at −20°C in single use aliquots</li>
</ul>
<p> </p>
<p>2. Permeabilization buffer (0.5% v/v Triton X-100 in 1XPBS)</p>
<ul>
<li>5 mL 10X PBS</li>
<li>45 mL nuclease-free water</li>
<li>250 μL Triton X-100 (Sigma; T8787)</li>
<li>Store at room temperature</li>
</ul>
<p> </p>
<p>3. 2X SSC buffer</p>
<ul>
<li>5 mL 10X PBS</li>
<li>45 mL nuclease-free water</li>
<li>250 μL Triton X-100 (Sigma; T8787)</li>
<li>Store at room temperature</li>
</ul>
<p> </p>
<p>4. 1X PBS buffer</p>
<ul>
<li>5 mL 10X PBS</li>
<li>45 mL nuclease-free water</li>
<li>Store at room temperature</li>
</ul>
<p> </p>
<p>5. 20% w/v Dextran sulfate</p>
<ul>
<li>In an RNase-free bottle (such as an empty nuclease-free water bottle), mix the following</li>
<li>100 g dextran sulfate (Fisher Scientific; bp1585100)</li>
<li>300 mL nuclease-free water</li>
<li>Stir with a stir bar cleaned with D/RNaseFree while heating gently on a hot plate (37 °C) until dissolved</li>
<li>Add nuclease-free water to produce a 500 mL total volume</li>
<li>Store at room temperature</li>
</ul>
<p> </p>
<p>6. Encoding probe hybridization buffer</p>
<ul>
<li>300 μL 100% deionized formamide (Ambion; AM9342)</li>
<li>500 μL 20% w/v dextran sulfate (above)</li>
<li>100 μL 20X SSC</li>
<li>90 μL nuclease-free water</li>
<li>1 mg yeast tRNA (Life Technologies; 15401-011)</li>
<li>10 μL 200 mM Vandyl Ribonucleoside Complex (VRC; New England Biolabs; S1402S)</li>
<li>Store at −20°C</li>
</ul>
<p> </p>
<p>7. Encoding probe wash buffer</p>
<ul>
<li>15 mL 100% deionized formamide</li>
<li>5 mL 20X SSC</li>
<li>29.5 mL nuclease-free water</li>
<li>0.5 mL 200 mM VRC</li>
<li>Store at 4°C in the dark and use within a few days</li>
</ul>
<p> </p>
<p>8. 40-mm diameter, No. 1.5 coverslips (Bioptechs; 40-1313-0319)</p>
<p>9. 60-mm diameter, Cell culture petri dish (Corning; 353802)</p>
<p>10. 1” x 3” microscope slides</p>
<p>11. Parafilm (VWR; 52858-076)</p>
<p>12. Orange FluoSphere carboxylate-modified beads, 0.1-um-diameter (ThermoScientific; F-8800)</p>
<p> </p>
<p> </p>
<p>This protocol requires the following equipment: </p>
<p> </p>
<p>1. Equipment for cell culture</p>
<p>2. 37°C incubator</p>
<p>3. 47°C incubator</p>
<p>4. Laboratory rocker</p>
<p style="text-align: justify;"> </p>

[STEPS]
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