Freezing Tissues for Cryosectioning

Histopathology Tissue Submission Freezing Tissues for Cryosectioning

Freezing Tissues for Cryosectioning

If you're not familiar with preparing tissues for cryosectioning or working with frozen sections, you should read
Cryotechniques for Light Microscopy © Woods and Ellis 2000.

There are many variables affecting the quality of frozen sections. You may have to experiment with different methods. Be advised that it can be difficult to get acceptable sections from some tissues. Fatty tissues are a notable example.

Also be advised that although we routinely use Plus (charged) slides to aid adherence of the section to the slide, one still must take care not to dislodge the sections during staining. Other means of increasing adhesion are available.

Embedding medium and molds
Most frozen tissues we receive are embedded in OCT in cryomolds. [Supplies page] Embedding medium helps conduct heat away from the specimen during freezing, helps protect the tissue from drying during storage, and supports the tissue during sectioning. We suggest placing a drop of OCT in the bottom of the mold and placing the tissue in the OCT. This will hold the tissue in place while you fill the mold with OCT. Just be careful to exclude large bubbles, fill the mold level full, and freeze by one of the methods below.

Label the mold with a lab marker before freezing.

If you want the tissue in a particular orientation, such as a piece of skin or hollow organ on edge, you can chill the filled mold, which will make the OCT thicken and help hold the tissue in place, but try to freeze the tissue as rapidly as possible to minimize freezing artifacts.

We also recommend not trying to embed large pieces of tissue, not more than about 1 cm x 1 cm x 4 mm. Don't crowd the mold with too many pieces of tissue.

Freezing methods
One simple method is to use dry ice (-70C) in block form placed in a styrofoam container. Place the filled cryomold on the block to freeze it. This method has the advantages of simplicity and safety, but does not freeze the tissue as rapidly as immersion in a freezing medium.

The method we prefer uses dry ice in pellet form. Place a small stainless steel bowl (or Pyrex or polypropylene beaker) in the bottom of a styrofoam container and fill the space around the bowl with dry ice pellets. Place some pellets in the bowl and slowly add isopentane (2-methyl butane) or acetone. Work in a fume hood, of course, as these are flammable. When the pellets stop bubbling vigorously, the "slurry" is ready. Once you've filled the mold and oriented the tissue, immerse it in the liquid to freeze it.

Isopentane also can be chilled in liquid nitrogen (-176C). With the liquid nitrogen in a styrofoam container or Dewar flask, use a tongs to lower a stainless steel, Pyrex, or polypropylene container of isopentane into the liquid nitrogen. The isopentane will start to become opaque as it nears freezing. Take the isopentane out of the liquid nitrogen and freeze the specimen as described above. Chill the isopentane again as necessary for subsequent tissues. This method has the advantage of very rapid freezing.

Frozen tissues can be stored in a -80 freezer. If the tissues were frozen in a flammable freezing medium, take care to allow it to evaporate before placing the blocks in the freezer.

Even if frozen in embedding medium, tissues must be protected from drying, which can ruin them. Tightly wrapped foil envelopes and screw-top plastic centrifuge tubes are commonly used, and it's a good idea to double wrap the specimens or place them in a container within a second container. Best, however, is to section and stain them as soon as possible.

See Cryotechniques for Light Microscopy © Woods and Ellis 2000 for more information.

Additional News and Announcements

  • UAB Institutional Signing Official Change Effective November 9, 2015

    Effective Monday, November 9, 2015, please use the following name and title on all OSP submissions for external sponsors requiring the name of the UAB Institutional Signing Official:
    Richard B. Marchase, Ph.D.
    Vice President for Research and Economic Development
     The remaining contact information, address, email and phone numbers, should remain the same:
    AB 1170
    1720 2nd Avenue South
    Birmingham, AL   35294-0111
    Phone:  205.934.5266
    Fax:  205-975-5977

    Any submissions in progress, in route or received by the OSP, will be updated to reflect the correct signing official.

    If you have any questions please contact the OSP at

    Posted by Office of Sponsored Programs | 11/05/15
  • Research Infrastracture Update

    IRAP - the Integrated Research Administration Portal - is an electronic tool that uses interconnected software modules to enhance each phase of the research process at UAB, from alerting investigators to new funding opportunities with the SPIN and SMARTS modules, to facilitating transfer agreements on research materials. Many documents can be shared across modules, eliminating the need to fill out redundant forms. Significantly, IRAP’s battery of more than 130 reports gives investigators granular data on the status of their submissions, so they always know where they are in the process. IRAP also allows administrators to improve workflows and procedures by finding and fixing bottlenecks in the system. In turn, this helps Research Administration offices work to deliver a better customer experience and to focus on improving customer service overall.
    Posted by Office of the Vice President for Research and Economic Development | 10/30/15
  • Notice of Proposed Rulemaking for the Federal Policy for Protection of Human Subjects

    ​A Notice of Proposed Rulemaking (NPRM) has been published seeking comments on proposed changes to the Federal Policy for the Protection of Human Subjects. The UAB Office of the IRB (OIRB) is requesting input from the UAB research community on the proposed changes in order to respond most effectively to the NPRM. The proposed rule includes significant changes to the regulations governing human subjects research. Among these changes are more relaxed requirements for certain categories of lower risk research and more stringent requirements for informed consent, especially in regard to use of biospecimens.
    Posted by Office of the Institutional Review Board for Human Use | 10/30/15
  • IRB Continuing Training Requirement for 2015

    ​UAB IRB policy requires continuing training once in every three year period for key personnel involved in human subjects research.  The UAB Office of the IRB has worked with the CITI Program to create different continuing training courses for (1) Biomedical and (2) Social/Behavioral/Educational researchers.  UAB personnel may complete either of these courses to meet continuing training requirements.  Both courses require investigators to complete three required modules and three of six elective modules. 
    Key personnel who completed their initial human subjects training before January 1, 2015 are required to complete one of the continuing training courses prior to December 31, 2015.  This continuing training will be valid for the 2015-2017 period.
    Additional information, including step by step instructions for registering, can be found on the OIRB website:  Continuing IRB Training.
    If you have questions about IRB training requirements, please contact the Office of the IRB at 934-3789.
    Posted by Office of the Institutional Review Board for Human Use | 10/09/15
  • New UAB Institutional Core Facility Program Launched

    The UAB Office of the Vice President for Research and Economic Development (OVPRED) announces the launch of the UAB Institutional Core Facility Program (ICFP), created to promote the development and operation of outstanding Core Facilities that can serve the needs of UAB investigators.
    Posted by Office of the Vice President for Research and Economic Development | 10/02/15
  • Change in State Law Regarding Age of Consent

    On May 7, 2015, Governor Bentley signed into law Act 2015-167, which lowered the age of consent to 18 years old (previously 19 years) for IRB-approved research conducted by an accredited college or university. This means that 18 year olds may now consent for themselves to participate in research conducted at UAB under IRB approval. The IRB has updated most of its forms with this information and will continue to revise policies and procedures to recognize this change in the law. The revised forms must be used for all submissions received in the OIRB after September 1, 2015.
    Posted by Office of the Institutional Review Board for Human Use | 06/26/15
  • IRB Form Changes - June 2015

    UAB IRB forms have been revised as of June, 2015. New versions of these forms will be required for all submissions received after September 1, 2015. These include: Human Subjects Protocol, Investigator's Progress Report, Sample Consent Form, Checklists for new submissions for both full and expedited reviews, Checklists for renewal submissions for both full and expedited reviews.
    Posted by Office of the Institutional Review Board for Human Use | 06/26/15
  • Confidential Disclosure Agreement Templates Updated

    UAB’s template confidential disclosure agreements have been updated and are available at the OSP Confidentiality Disclosure Agreement (CDA) webpage. The following confidential disclosure agreement (CDA) templates are available:

    Mutual Confidential Disclosure Agreement (Company and UAB disclosing confidential information);

    Unilateral Confidential Disclosure Agreement (Company Disclosing to UAB); and

    Unilateral Confidential Disclosure Agreement (UAB Disclosing to Company)

    Please use, and encourage the contracting party to use, the appropriate UAB template CDA. If the contracting party can agree to the terms of one of UAB’s template CDAs, UAB will sign the agreement without further routing and review, resulting in a more expeditious process.

    Please forward any questions regarding the CDA process, or UAB’s template CDAs to

    Posted by Office of Sponsored Programs | 06/22/15
  • Policy Update: Definition of a Principal Investigator

    Effective May 18, 2015 UAB’s Definition of a Principal Investigator policy is modified with regard to UAB employees in the 02 (Full-time Temporary) employment status or other UAB employment categories. See the full policy at the link or via the UAB Policies & Procedures Library.

    Case-by-case inquiries should be addressed to
    Posted by Office of Sponsored Programs | 06/01/15
  • Expired Form FDA 1572 continued use approved

    ​On April 30, 2015 the Form FDA 1572 on the FDA website at is expired.

    The FDA is in the process of updating and posting a new Form FDA 1572. Even though it is expired, the FDA approved continued use of the current form.

    We will monitor the FDA website and will post notice of the new form's release, when available.

    Posted by Office of the Institutional Review Board for Human Use | 05/06/15
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