This article details the process of cryopreserving murine brain samples through snap freezing techniques. The method ensures the retention of brain architecture for subsequent histological analysis.
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 any traces of sucrose.
Next, position the brain in the desired orientation into a deep cryomold containing a layer of embedding medium.
Overlay the brain in the cryomold with more embedding media to ensure the entire organ is covered.
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.
Now, allow the brain specimen to freeze until the embedding medium solidifies forming a gelatinous layer around the brain, holding it in place.
Once frozen, remove the cryomold from the isopentane bath and wrap the cryopreserved mouse brain in a foil sheet.
Store the sample at minus eighty degrees Celsius for further histological analysis.
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.
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.
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.
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