Freezing Tissues for Cryosectioning

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

  • 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
  • White Paper Solicitation for Major New Alabama Research Initiatives

    ​Alabama EPSCoR (ALEPSCoR) would like to solicit White Papers for major new research initiatives in targeted areas where Alabama can achieve national competitiveness and leadership. The goal of these targeted research initiatives will be to create the necessary State-wide research and education infrastructure to foster ground-breaking technology innovations resulting in substantial federal grant funding and intellectual property development that will create jobs for the State. The desired outcome of this white paper solicitation is to select one or two major new research initiatives for future investment and large scale multi-institutional proposal submissions.
    Posted by Office of the Vice President for Research and Economic Development | 09/16/15
  • Dr. Rakesh Patel, Ph.D. announced as new Director for the Center for Free Radical Biology

    Dr. Selwyn Vickers, M.D., F.A.C.S, Senior Vice President for Medicine and Dean, School of Medicine, and Dr. Richard Marchase, Ph.D., Vice President for Research and Economic Development, are pleased to announce that Rakesh Patel, Ph.D., will be the new director of the Center for Free Radical Biology (CFRB) from October 1, 2015, when Dr. Victor Darley-Usmar, Ph.D. steps down after serving as Director since 2006.
    Posted by Office of the Vice President for Research and Economic Development | 09/10/15
  • 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
  • Additional OSP Dropbox Enhancements

    The Office of Sponsored Programs is pleased to announce additional enhancements to the OSP dropbox for electronic SF424 grant applications to The link to access the OSP dropbox is OSP Drop Box for Electronic Submissions to . The revised OSP dropbox user guide can be found at OSP Dropbox Quick Submission Guide. New enhancements to the dropbox include:

    • The attribute page has been modified to prompt the user to create a record and then upload the files. When the proposal record is created the attribute page is refreshed with the comment “Submit Successful*” with a footnote to continue to upload files (“*Please continue on to the Upload Files section and attach your proposal documents”).

    • Once the proposal record is created, the Upload file tab is now used to upload the proposal documents.

    • The upload file history can now be viewed using the “File History” section.  This allows users to verify that the upload was successful.

    If you have any questions regarding the new OSP dropbox functionality, please contact your OSP federal officer or

    Posted by Office of Sponsored Programs | 04/21/15
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