Name: Flash Freezing and Cryosectioning E12.5 Mouse Brain (Video) | JoVE Journal
Uploaded: 2007-05-28T00:00:00.000+00:00
Duration: 28 min 32 s
Description: Learn cryosectioning techniques for neuroscience research using E12.5 mouse embryonic brain tissue. This method demonstrates flash freezing, OCT embedding, and troubleshooting tips to obtain distortion-free sections for in situ hybridization and immunofluorescence.

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<ol>
<li>Fix tissue in 4% paraformaldehyde in PBS for desired time.</li>
<li>Sucrose infuse tissue (cryoprotection) <ol>
<li>Make 30% sucrose solution in PBS w/v in 2059 tube.</li>
<li>Rinse tissue 3x in PBS (~ 5 min with rocking).</li>
<li>Place tissue in 30% sucrose solution.&nbsp; Tissue will not sink.</li>
<li>Place the tissue in 4&deg;C overnight, or until it has sunk.</li>
</ol></li>
<li>Label appropriate size cryomold with information and orientation.</li>
<li>Fill cryomold with O.C.T. (avoid bubbles).</li>
<li>Transfer tissue to O.C.T. bath and coat it with O.C.T.</li>
<li>Transfer tissue to O.C.T. in cryomold.</li>
<li>Orient the tissue under microscope.</li>
<li>Pour liquid nitrogen into plastic Petri dish.</li>
<li>Quickly and carefully lower the tissue in cryomold into the nitrogen. (do not submerge the top of the cryomold.)</li>
<li>When the O.C.T. is solid white, place the frozen tissue into -80&deg;C freezer for storage.</li>
<li>Equilibrate tissue to ~20&deg;C for at least 30 min. prior to sectioning.</li>
</ol>

<table><tbody><tr><td>Tissue-Tek Cryomold</td><td>Ted Pella, Inc.</td><td>27181</td><td></td></tr><tr><td>O.C.T.</td><td>Ted Pella, Inc.</td><td>27050</td><td></td></tr><tr><td>Sucrose solution</td><td></td><td></td><td>30% sucrose solution in PBS w/v</td></tr><tr><td>paraformaldehyde</td><td></td><td></td><td>4% paraformaldehyde in PBS </td></tr></tbody></table>

flash freezing and cryosectioning e12.5 mouse brain

Flash Freezing and Cryosectioning E12.5 Mouse Brain

<p class="jove_content">Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse.  Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.</p>

<p>Hi, I'm Spencer Curley from University of California Irvine and Ed Maki's Lab, and I am going to demonstrate how to do cryosectioning. To do that, I use these commonly used reagents first. The embedding compound or embedding media that I use is called OCT for optimal cutting temperature.</p><p>It's a clear solution, it's very thick and viscous, and it holds the tissue nicely while you can orient it and then it freezes, and ultimately it's water soluble so you can get rid of it during any type of staining procedure. So I embed the tissue in a mold. These cryo mold biopsy containers, they come in various sizes.</p><p>I will be using this smaller mold here because I do embryonic tissue from mouse. We flash freeze our tissue and liquid nitrogen. So first I'm gonna fill this protective vacuum flask with liquid nitrogen from our tank here.</p><p>Prior to flash freezing the tissue, you have to what's called cryo protect the tissue, otherwise freezing itself will destroy the tissue. So what you do is you sink the tissue in a 30%sucrose solution overnight. And if it takes longer, that's all right, just as long as the tissue sinks.</p><p>And as you can see, visa embryonic day 12.5 mouse heads are, have sunk in the, in </p>

flash-freezing-and-cryosectioning-e125-mouse-brain

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Learn cryosectioning techniques for neuroscience research using E12.5 mouse embryonic brain tissue. This method demonstrates flash freezing, OCT embedding, and troubleshooting tips to obtain distortion-free sections for in situ hybridization and immunofluorescence.

Flash Freezing and Cryosectioning E12.5 Mouse Brain

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