How do I harvest and preserve fresh frozen tissue for sectioning?

Proper handling and preservation of tissue is essential for maintaining tissue and RNA quality

This document describes guidelines for harvesting and preserving fresh murine brain for -18 C cryostat sectioning and Seeker Tile analysis. It may not reflect the best
conditions for your tissue type. Please use your own protocol and conditions ideal for your tissue type.

Materials

All materials, reagents, consumables and equipment are suggested and can be replaced by equivalent materials.
Required Reagents and Consumables

Equipment

● Styrofoam container
● Gloves for handling dry ice
● Metal bowl for isopentane bath
● Tweezer or forceps
● Supports for plastic molds while sitting in isopentane bath

Guidelines:

A. Preparation before harvesting

Isopentane Bath

1. Place dry ice or liquid nitrogen in a safe container and create a flat surface.
2. Depending on the size of your tissue and the plastic mold you are using, place an
appropriate amount of isopentane in a metal container and place it on the flat
surface.
3. Fill the area around the isopentane container with dry ice or liquid nitrogen. Ensure
the dry ice or liquid nitrogen outside of the metal isopentane container is level with
the amount of isopentane.
4. Place two supports on the edge of the isopentane bath so that the plastic mold
can rest on the supports while in the isopentane bath without exposing the tissue
and the OCT to isopentane.
5. Prepare and label a plastic mold for each tissue for embedding.
6. Place a bubble free layer optimal cutting temperature compound (OCT) in the
bottom of each mold and leave at room temperature.
7. Prepare a cold bath of 1X PBS for washing tissue after dissection.
8. Optional: Depending on your tissue of interest, consider anesthetizing and whole
animal perfusion with ice cold HEPES-Sucrose Cutting Solution.
(https://www.protocols.io/view/fresh-frozen-mouse-brain-preparation-for-single
-nu-j8nlkedb1l5r/v1)

Dry Ice only

1. Place dry ice in a styrofoam container and create a flat surface.
2. Prepare and label a plastic mold for each tissue for embedding.
3. Place a bubble free layer optimal cutting temperature compound (OCT) in the
bottom of each mold and leave at room temperature. .
4. Prepare a cold bath of 1X PBS for washing tissue after dissection.

B. Sample collection and handling

1. Clean freshly harvested and trimmed tissue in a bath of cold PBS to wash off any
blood, hair and loose tissue.
2. Place the tissue on kimwipes and gently blot dry.
3. Immediately place freshly harvested tissue into pre-prepared plastic molds with
OCT at the bottom.
4. Carefully cover the fresh tissue with bubble free OCT.
5. Once the tissue is fully embedded in OCT, rest the plastic mold on
a. the flat dry ice surface.
b. the supports to ensure the mold is partially submerged in the isopentane
but the OCT and tissue do not come in contact with the isopentane.
6. Let freeze for a few minutes depending on the size and volume of your molds.
7. Once the OCT and embedded tissue are completely frozen, place the mold in a
plastic bag and store the block in the -80 degrees Celsius freezer for one day
before further processing.

C. Sectioning tissue

8. After one day in the -80 degrees Celsius freezer, remove the block from the mold
and trim the excess OCT from the block.
9. Mount the block onto a cold chuck from the cryostat at -18 degrees Celsius by
adding a layer of OCT to the chuck and pressing the block in the desired
orientation into the fresh OCT.
10. Place the chuck with the mounted block into the cryostat with the tissue resting on
the chuck for freezing and complete mounting.
11. Leave the block to acclimate to the cryostat for at least 20 minutes.
12. Once the chuck is frozen to the block, fix the chuck into the cryostat and bring the
cryostat to -18 degrees Celsius.
13. Set the cryostat to cut 10 μM sections.
14. Once acclimated, section the tissue until the blade has reached your region of
interest.
15. Section 10 μM sections of your tissue at the region of interest.

Please note that optimum cutting temperature and acclimation time will depend on
your tissue type and block size.

16. Proceed to mount sections on a slide for H&E staining (See Guidelines: H&E Staining
for Fresh Frozen Tissue) and/or Seeker Tile analysis (Seeker: User Manual for Fresh
Frozen Tissue).
17. When finished sectioning, label a plastic bag and place the remaining chuck and
tissue block in the plastic bag.
18. Place the labeled bag back with chuck and tissue in it into the -80 degrees Celsius
freezer for storage.