The cryosection procedure is also known as the frozen section procedure. It is a laboratory procedure used to perform microscopic analysis of a specimen. This procedure is most commonly used in oncological surgery. A pathologist is necessary for the intraoperative consultation. The pathologist evaluates and examines the specimen. The pathologist then reports if the specimen is benign or malignant. They are also responsible for informing if the resection margin is clear of cancer. The cryosectioning procedure practiced today is based on the technique described by Dr. Louis B. Wilson in 1905. Cryosections are a good way to visualize the fine details of the cell. Although less stable than resin and paraffin-embedded sections, cryosections are typically more superior for the preservations and detection of antigens through microscopy. Cryosection preparation can usually be done in a day. The rapid freezing helps decrease the formation of ice crystals and minimizes morphological damage. Cryosections can be used in various procedures such as in situ hybridization, immunohistochemistry, and enzymatic degradation.
Some of the reagents used in a cryosectioning protocol are:
· Fixative (such as formaldehyde)
· Optimal cutting temperature (OCT) compound (such as Tissue-Tek; Sakura Finetek USA)
· Staining solution (such as toluidine blue, hematoxylin, eosin, or others)
· Fresh tissue sample
Important equipment that would be required in a cryosectioning protocol are:
· Brush (camel hair)
· Container for storage of tissue sample
· Cryostat with metal grids (The cryostat can be likened to a microtome in a freezer. It is a machine capable of slicing thin sections. Although expensive, hospital pathology laboratories may have cryostats available for rental)
· Microscope slides (The slides can be silanized or poly-L-lysine coated)
· Plastic or metal tissue mold
· Moistened tissues
The procedure for cryosectioning can be done quickly as it is relatively simple.
A) Cryostat preparation
i) Use proper cleaning agents and clean the cryostat.
ii) Insert a new sterile blade for the cryostat.
iii) When it comes to frozen embedded block or fresh frozen specimens, use some frozen embedding media to adhere the sample to the mount in the proper cutting position. Ensure that the cutting surface is parallel to the blade.
iv) Allow the specimen to equilibrate reaching cryostat temperature. This takes approximately 20 minutes.
B) Cryosection preparation
i) Freeze a tissue sample up to 2.0 cm in diameter in OCT using a suitable tissue mold. Freeze the OCT with tissue onto the metal grids fitting the cryostat. At room temperature, OCT is viscous but freezes at -20⁰C. Depending on the type of tissue, optimal freezing temperature may differ. For example, brain tissues are optimally frozen at -3⁰C in M-1 medium.
ii) Cut sections that are approximately 5 to 15μm in the cryostat at a temperature of -20⁰C. the temperature of the cutting chamber can be adjusted based on the tissue under study. These sections can be moved with the help of toothpicks and brushes if necessary. A camel hair brush can be helpful in guiding the merging section over the blade.
iii) Remove the folds and wrinkles present on the cut sections.
iv) Within 1 minute of cutting the tissue section, transfer it to a room temperature slide by touching the slide to the tissue. This allows the cut section to adhere to the slide. Using your gloved finger, rub the underside of the slide to help transfer heat as this helps with the adhesion. All this should be accomplished within 1 minute of cutting the section. This avoids the freeze drying of the specimen. Using silanized or poly-L-lysine coated slides improve the adherence of the section.
v) An optional step is to use ultraviolet treatment of the slide to increase the adherence and with sterilization. This can be done by incubating the slide for 15 to 20 minutes under the ultraviolet light. It works best if an ultraviolet sterilization hood is utilized. The light breaks down the membrane slightly helping with adhesion and sterilization. It is important to not incubate it longer than 30 minutes as it risks damaging the membrane.
vi) To evaluate the preservation and orientation of the tissue, the first slide of each set can be stained using toluidine blue, eosin, hematoxylin, or various aqueous stain.
vii) Immerse the slide immediately into a fixative. Some researchers air-dry the section onto the slide at air temperature to maximize adherence before fixation. However, this technique has a disadvantage where the surface tension forces distortion of the cells resulting in the loss of high-resolution detail. It can also lead to changes in the results of immunostaining.
viii) Any unused tissue should be covered using a layer of OCT to avoid freeze drying and storing leftover samples at -70⁰C. For long-term storage, adding moistened tissue to the container helps prevent desiccation especially in a frost-free freezer.
If the tissue is difficult to section, it is important to consider the following:
· If the frozen tissue in OCT is not cut in a thin and smooth sheet, the knife could be dull.
· The tissue can be difficult to section if the tissues have variable textures, water, or fatty.
1) Fischer AH, Jacobson KA, Rose J, Zeller R. Cryosectioning tissues. CSH Protoc. 2008: pdb.prot4991.
2) Protocol Cryosectioning. Molecular Machines & Industries. Accessed 1/8/2019. http://www.molecular-machines.com/libraries.files/Cryosectioning.pdf