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

Abstract

Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion. J. Vis. Exp. (2020)

This video demonstrates the cryopreservation of a mouse’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.

Protocol

1. Cryopreservation of Brains Harboring Glioma Tumors

1. 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.
2. Remove the brain from the 30% sucrose solution and blot it dry on a filter paper.
3. 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.
4. 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.
5. Once the OCT solidifies (~30–40 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 °C.

Materials

Peel Away Disposable Embedding Molds	Electron Microscopy Sciences	70182
Research Cryostat	Leica	CM3050s
Tissue-Plus O.C.T. Compound	Fisher Scientific	23-730-571