Freezing Tissues for Cryosectioning

Histopathology Tissue Submission Freezing Tissues for Cryosectioning

Freezing Tissues for Cryosectioning

Background
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.

Storage
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

  • Office of Sponsored Programs Announcement - New Website Launch

    The Office of Sponsored Programs is pleased to announce the launch of our new OSP website. The new OSP website offers users a more efficient navigational design structure with enhanced content categories.

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    For additional information, please contact Mike Matthews at mimatt@uab.edu or Jonathan Price at jonprice@uab.edu.

    Posted by Office of Sponsored Programs | 04/21/16
  • Center for Disability Health and Rehabilitation Science Guest Lecturer: Dr. Robert Motl

    Center for Center for Disability Health and Rehabilitation Science presents

    Professor Robert W. Motl, PhD
    Department of Kinesiology and Community Health Neuroscience Program
    University of Illinois at Urbana - Champaign: 

    Physical Activity and Multiple Sclerosis - What We’ve Learned and Where We Need To Go

    Thursday, April 14, 2016 12:00-1 p.m.
    School of Health Professions Building Executive Learning Center, Room 640

    Bring Your Lunch

    Beverages will be provided

    To RSVP, please contact Jane Moore at (205) 934-5909 or Email: djmoore@uab.edu  no later than Noon Wednesday 4/13

    Posted by Center for Disability Health and Rehabilitation Science | 04/12/16
  • Call for Abstracts: 7th Annual UAB Diabetes Research Day

    ​Registration is now open for the 7th Annual UAB Diabetes Research Day to be held on May 3, 2016 at the Doubletree Hotel.  Please join us for this event where researchers from across campus and other institutions present their diabetes related research via oral presentations and poster presentations.

    Abstracts Due Monday, April 11, 2016.

    Abstract Guidelines:
    For abstract submission, please include a title, the full name of all authors, affiliations (Division, Department, University) and underline the presenting author’s name. The body of the abstract should be less than 250 words. Please use Arial 11 font for all text. Email abstracts in a word file to mpreuss@uab.edu. If you are a trainee/staff, please copy your PI on the abstract submission.
     
    There is no cost for attendance, but registration is required to reserve lunch. Please register by April 25th!
    Posted by Comprehensive Diabetes Center | 04/06/16
  • NIH eSubmission Items of Interest - March 25, 2016

    The NIH Grants and Funding website has been redesigned and simplified with a video tour available. FORMS-D Application Instructions are now available. FORMS-D Application Packages are now available for some FOAs. FORMS-C should be used for applications with due dates on or before May 24, 2016. FORMS-D should be used for applications with due dates on or after May 25, 2016.
    Posted by Office of Sponsored Programs | 03/25/16
  • NIH eSubmission Items of Interest - February 29, 2016

    NIH eSubmissions Items of Interest for February 29, 2016. FORMS-D application changes are on the way. FORMS-D must be used for applications with due dates on March 25, 2016 or after. Read full article for a preview of FORMS-D and guidance on determining when and how to use FORMS-D.
    Posted by Office of Sponsored Programs | 03/01/16
  • Reminder Regarding ASSIST Grant Applications

    As the February 5th deadline for new NIH R01 submissions approaches, OSP would like to remind you that all competing, single-project NIH grant applications can now be submitted through ASSIST.
    Posted by Office of Sponsored Programs | 01/25/16
  • Request for Nominations for Director of the Center for Structural Biology (CSB)

    ​The UAB CSB Internal Search Committee invites nominations of UAB faculty members for the position of Director of the Center for Structural Biology (CSB).  The CSB was established in 2011, and is one of 24 University-wide Interdisciplinary Research Centers (UWIRCs); it has been led by Lawrence J. DeLucas, Ph.D., since its inception.  The mission of the Center for Structural Biology is to support research and education through exploration of structure/function relationships for biological macromolecules and the development of novel technologies for biomedical research which enhance UAB’s basic and translational research goals.  Center membership includes more than 125 faculty and non-faculty members and hosts a number of on-site core facilities, as well as access to a national laboratory x-ray beam.
     
    The Director will oversee all of the interdisciplinary center activities, be a member of the Council of Center Directors and be responsible for interfacing with participating schools and managing the budget.  Nominations, including self-nominations, should include a CV and a one (1) page summary outlining the qualifications of the proposed Director and summarizing ideas and plans if appointed to the position.  The decision will be made after review and interviews by the Search Committee.
     
    Nominations should be submitted to Jamie Seitz (jbseitz@uab.edu) by Friday, January 29, 2016. 
    Posted by Office of the Vice President for Research and Economic Development | 01/19/16
  • MTO Change in Process for Finalized Agreement Documents

    The Material Transfer Office is changing the process for providing finalized agreements to the campus. Previously the finalized documents were emailed to the investigator, now the investigator will be notified that the finalized agreement is available for viewing through an eReport.

    Details can be found on the IRAP Website.

    Posted by Office of the Vice President for Research and Economic Development | 12/10/15
  • 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:
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    Vice President for Research and Economic Development
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    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 askosp@uab.edu.

    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
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