<p class='jove_title'><strong>1. Cryopreservation of Brains Harboring Glioma Tumors</strong></p>
<ol>
	<li>Prior to cryopreserving the brains, prepare a jar filled with cold isopentane/2-methylbutane and place it into a container filled with liquid nitrogen. Let the solvent cool down.</li>
	<li>Remove the brain from the 30% sucrose solution and blot it dry on a filter paper.</li>
	<li>Label the cryomold with a permanent marker. Carefully add approximately 5 mL of OCT (optimal cutting temperature compound) into the center of the cryomold avoiding air bubbles.</li>
	<li>Place the brain into a cryomold containing OCT in the desired orientation. Fill the mold with OCT until the brain is fully submerged. Using clean forceps, quickly place the cryomold with OCT and the brain into the cold isopentane/2-methylbutane.</li>
	<li>Once the OCT solidifies (~30&#8211;40 s), remove the cryomold with the brain and place it in dry ice. Do not leave the mold containing the brain in 2-methylbutane past 2 min as this may cause cracks in solid OCT. Wrap the cryomold with the brain in aluminum foil and store it at -80 &#176;C.</li>
</ol>

<table><tbody><tr><td>Peel Away Disposable Embedding Molds&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>70182</td><td></td></tr><tr><td>Research Cryostat&amp;nbsp;</td><td>Leica&amp;nbsp;</td><td>CM3050s</td><td></td></tr><tr><td>Tissue-Plus O.C.T. Compound&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific</td><td>23-730-571</td><td></td></tr></tbody></table>

cryopreservation of murine brain by snap freezing: a technique to rapidly freeze whole mouse brain specimen for histological analysis

Cryopreservation of Murine Brain by Snap Freezing: A Technique to Rapidly Freeze Whole Mouse Brain Specimen for Histological Analysis

<p class='jove_content'>Source: Comba, A. <em>et al.</em> <a href='https://www.jove.com/v/60939/laser-capture-microdissection-glioma-subregions-for-spatial-molecular'>Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion.</a> <em>J. Vis. Exp.</em> (2020)</p>
<p class='jove_content'>This video demonstrates the cryopreservation of a mouse&#8217;s brain to isolate discrete anatomical areas or specific cell populations from the tumor tissues. This technique is critical in obtaining optimal optical resolution of brain tissue morphology for subsequent analysis.</p>

Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, ...

<p>To cryopreserve murine brain sample by snap freezing, begin by taking a beaker filled with isopentane solution - a highly conductive liquid - and immerse it into a jar containing liquid nitrogen.</p>
<p>Freeze the isopentane till the solution turns viscous.</p>
<p>Meanwhile, take a whole mouse brain sample prefixed in sucrose solution. This treatment prevents brain tissue deformation in the subsequent steps.</p>
<p>Remove the brain sample from the sucrose solution and blot it dry to remove any traces of sucrose.</p>
<p>Next, position the brain in the desired orientation into a deep cryomold containing a layer of embedding medium.</p>
<p>Overlay the brain in the cryomold with more embedding media to ensure the entire organ is covered.</p>
<p>Then, submerge the cryomold containing the embedded brain into the pre-chilled isopentane bath. The rapid cooling of the brain tissue to an ultra-low temperature ensures retention of brain architecture without any distortion.</p>
<p>Now, allow the brain specimen to freeze until the embedding medium solidifies forming a gelatinous layer around the brain, holding it in place.</p>
<p>Once frozen, remove the cryomold from the isopentane bath and wrap the cryopreserved mouse brain in a foil sheet.</p>
<p>Store the sample at minus eighty degrees Celsius for further histological analysis.</p>

<p>To prepare for the cryopreservation, fill a jar with cold isopentane/2-methylbutane and place the jar into a container filled with liquid nitrogen. While the solvent is chilling, blot the brain dry on a piece of filter paper and place the brain into a labeled cryomold.</p>
<p>Carefully add approximately 5 milliliters of optimal temperature cutting medium to the center of the cryomold and place the brain into the mold in the desired orientation. Using clean forceps, quickly place the cryomold into the chilled isopentane/2-methylbutane for 30 to 40 seconds.</p>
<p>Once the cutting medium has solidified, transfer the cryomold onto dry ice and wrap the cryomold and snap-frozen brain tissue in aluminum foil for minus 80 degrees Celsius storage.</p>

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6.2K Views. To cryopreserve murine brain sample by snap freezing, begin by taking a beaker filled with isopentane solution - a highly conductive liquid - and immerse it into a jar containing liquid nitrogen. Freeze the isopentane till the solution turns viscous. Meanwhile, take a whole mouse brain sample prefixed in sucrose solution. This treatment prevents brain tissue deformation in the subsequent steps. Remove the brain sample from the sucrose solution and blot it dry to remove an...

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<table><tbody><tr><td>Peel Away Disposable Embedding Molds&nbsp;</td><td>Electron Microscopy Sciences</td><td>70182</td><td></td></tr><tr><td>Research Cryostat&nbsp;</td><td>Leica&nbsp;</td><td>CM3050s</td><td></td></tr><tr><td>Tissue-Plus O.C.T. Compound&nbsp;&nbsp;</td><td>Fisher Scientific</td><td>23-730-571</td><td></td></tr></tbody></table>

Cryopreservation of Murine Brain by Snap Freezing: A Technique to Rapidly Freeze Whole Mouse Brain Specimen for Histological Analysis

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