Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

Overview

In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Sectioning Frozen Brain Tumor Tissues

1. Label 2 µm polyethylene naphthalate (PEN) slides with the sample information. Tissue sections will be placed directly on these slides following sectioning.
2. Set the temperature of the cryostat chamber between -20 to -24 °C. Before sectioning, place the sample block in the cryostat chamber and let it equilibrate to the temperature in the chamber for 30–60 min.
3. Clean the cryostat chamber and the knife holder with 100% ethanol and spray the brushes to be used with RNase cleaning solution. Working inside the cryostat chamber, remove the mold and attach the OCT block containing the brain to the cryostat specimen disk with OCT. Place the block in the disk holder and align the block with the knife blade.
4. Install a disposable blade into the sectioning holder.
5. Section the brain at 10 µm thickness. Make sure there are no streaks or scratch lines in the tissue. Using a paintbrush, cautiously flatten and uncurl the tissue onto the cutting surface.
6. Carefully mount the tissue containing the brain sections onto RNase-free PEN glass slides. Flip the positively charged glass slides with the fingers in direction of the tissue and smoothly press the glass slide down towards the tissue section.
   NOTE: Temperature of hands will help the tissue attach to the glass.
7. After mounting the brain sections onto the slides, keep the slides in a box inside the cryostat chamber and then store them at -80 °C. Never keep the slides at room temperature.
   NOTE: Folding of the tissue and tearing are common. For accurate posterior analysis, it is important to minimize these artifacts.

Materials

Name	Company	Catalog Number	Comments
PEN Membrane Glass Slide (2 µm)	Lieca	1150518
Research Cryostat	Leica	CM3050s
RNaseZap RNase Decontamination Solution	Fisher Scientific	AM9780
Peel Away Disposable Embedding Molds	Electron Microscopy Sciences	70182
Pinpoint Solution	Zymo Research	D3001-1
Tissue-Plus O.C.T. Compound	Fisher Scientific	23-730-571