Experiments in the preparation of frozen tissues and sections

Summary

For better results, fresh tissue should be used, because samples stored at -80°C for more than 24 h will have much lower RNA yield and integrity. Although several experimental groups have reported obtaining undegraded RNA from paraffin-embedded tissues using the LCM technique, the authors recommend the use of frozen tissue blocks. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

Experiments in the preparation of frozen tissues and sections

Materials and Instruments

Fresh Tissue
Acetone Distilled water Dry ice Phosphate buffer (PBS) Tek OCT Tissue Compound
Closed containers Beakers Constant-cooling devices Dissection tools Flat plates Tissue molds Slides Blades

Move

I. Materials

1. Buffers, solutions and reagents

Acetone

Distilled water

Dry ice

Phosphate buffer solution (PBS)

Tek OCT tissue complex (Sakura FinetekUSA, Torrance, CA)

2. Specialized equipment

Closed containers

Beakers

Cooling devices

Dissection tools

Flat plate

Disposable tissue mold that can be chipped (Pelco International, Redding, CA)

Plain, non-adhesive slides

Sakura blades (IMEB, SanMarcos, CA)

3. Cells and tissues

Fresh tissue (see (2) in II. Methods)

II. Methods

1. Preparation of frozen tissue

(1) Cover the mold containing the embedding medium (Tek O C T Tissue Complex is recommended) and prepare a dry ice-acetone bath (simply mix dry ice and acetone in a beaker).

(2) Execute the animal and remove the destination tissue to mix with the embedding medium.

(3) Place the tissue on the bottom of the cold mold. To facilitate the utilization of the constant-cooling device, the destination section is placed on the bottom of the cold mold in reverse.

(4) Place the bottom of the cold mold on the surface of a dry ice-acetone bath so that the sample will cool gradually within 3~4 min. The product will appear white when frozen. Do not drop the mold into the acetone while freezing.

(5) Remove the tissue from the mold and place at -80°C until sectioning. If tissue samples are left for a longer period of time, place them in an airtight container with cold distilled water to maintain humidity.

2. Slicing and Preservation

(1) Take frozen samples and place them in a cryogenic cooler containing O C T complex, using the standard frozen tissue sectioning method.

(2) Allow the sample to equilibrate with the temperature in the cold box (-25~-20°C) for 15~20 min. The equilibration time can be extended moderately if the tissue block is too cold when the sample is sectioned.

(3) Place the sections (1~5um thickness) intended for LCM on a plain, non-adhesive slide. If the tissue sticks too tightly to the slide, it may not be favorable for subsequent LCM operations.

(4) Place on dry ice immediately after placement. If LCM is done on the same day, the slides should be placed on dry ice. Otherwise, they should be stored at -80°C.

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