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

  • NIH Notices – August 22, 2016

    Notices

    • New Policy Eliminates Most Appendix Material for NIH/AHRQ/NIOSH Applications Submitted for Due Dates On or After January 25, 2017
      (NOT-OD-16-129) National Institutes of Health
      Agency for Healthcare Research and Quality
      National Institute for Occupational Safety and Health
    • Changes to the NIH/AHRQ Policy on Post-Submission Materials for Applications Submitted for Due Dates On or After January 25, 2017
      (NOT-OD-16-130) National Institutes of Health
      Agency for Healthcare Research and Quality
      National Institute for Occupational Safety and Health
    • Projected FY 2017 Stipend Levels for Postdoctoral Trainees and Fellows on Ruth L. Kirschstein National Research Service Awards (NRSA)
      (NOT-OD-16-131) National Institutes of Health
      Agency for Healthcare Research and Quality
      Health Resources and Services Administration
    • Revised: Projected FY 2017 Stipend Levels for Postdoctoral Trainees and Fellows on Ruth L. Kirschstein National Research Service Awards (NRSA)
      (NOT-OD-16-134) National Institutes of Health
    Posted by Office of Sponsored Programs | 08/22/16
  • UAB Joins Federal Demonstration Partnership’s (FDP) Clearinghouse Pilot Program

    In an effort to streamline the process for both incoming and outgoing federal subawards, UAB has joined the Federal Demonstration Partnership’s (FDP) Expanded Clearinghouse. This is a pilot program for certain FDP institutions and provides audit, demographic and fiscal information needed by pass-through entities (PTEs) when they are issuing federal subawards. Now, when UAB issues a federal subaward to another entity in the pilot program, collection of the Subrecipient Assurance form is no longer required. Instead, the necessary information will be obtained from the entity’s profile in the Expanded Clearinghouse; a searchable list of the participating entities along with their profiles can be found FDP Expanded Clearinghouse website.

    Attached is a PDF of the participating institutions. Project-specific information (such as Human Subjects use, Animal Subjects use, etc.) will still need to be collected from the entities including a new, project specific form for that will be released shortly.

    For incoming subawards, we will no longer need to complete forms containing this information from other pilot institutions. The FDP web site has sample language to send to other pilot participants when we are asked to complete such forms.

    Additional information can be found on the FDP website and will be available shortly the Office of Sponsored Programs website. If you have questions, please contact askosp@uab.edu.

    Posted by Office of Sponsored Programs | 08/22/16
  • Conflict of Interest Re-training Required for Many Investigators this Year

    On August 24th 2012, financial conflict of interest training became mandatory for all research personnel responsible for the design, conduct or reporting of research. The Federal Final Rule that established this requirement also stipulates that re-training is required every four years. For many investigators, re-training will be required this year. Email notifications will be sent out informing investigators when they need to re-train. Please contact the Office of the Conflict of Interest Review Board (OCIRB) at (205) 975-9691 if you have any questions about re-training.

    Posted by Office of the Conflict of Interest Review Board | 08/22/16
  • Department of Defense Breast Cancer Research Program Funding Opportunities for Fiscal Year 2016

    Per the Fiscal Year 2016 (FY16) Defense Appropriations Act, $120 million was allocated to the Department of Defense Breast Cancer Research Program (BCRP) in support of breast cancer research. FY16 BCRP Program Announcements and General Application Instructions for the following award mechanisms are posted on the Grants.gov.
    FY16 BCRP applications must address one or more of the following challenges:
    • Prevent breast cancer (primary prevention)
    • Identify determinants of breast cancer initiation, risk, or susceptibility
    • Distinguish deadly from indolent breast cancer
    • Conquer the problems of overdiagnosis and overtreatment
    • Identify what drives breast cancer growth; determine how to stop it
    • Identify why some breast cancers become metastatic
    • Determine why/how breast cancer cells lay dormant for years and then re-emerge (recurrence); determine how to prevent recurrence
    • Revolutionize treatment regimens by replacing them with ones that are more effective and less toxic
    • Eliminate the mortality associated with metastatic breast cancer
    Please review the attached announcement (PDF) for more detailed information and instructions regarding applications.
    Posted by Office of Sponsored Programs | 08/22/16
  • Research Town Hall: The Implications of the FLSA Mandate on Postdoc Salaries

    ​​Join us on Wednesday, August 24 in Spain Auditorium at 4 pm for an open discussion on the implications of the FLSA mandate on postdoc salaries.  Deans and Central Administration are investing in a plan that we see as striving to be fair to all our postdocs, while at the same time easing the burden on our investigators in order to minimize disruption to ongoing research programs. We will also discuss the Research Career ladder and its implementation.  Event information can be found here.

     

     

    Posted by Office of the Vice President for Research and Economic Development | 08/18/16
  • eRA Enhancements - August 17, 2016

    Expect new features and improvements to be released on August 18, 2016, for the Extramural Trainee Reporting and Career Tracking (xTRACT) system. 

    Features

    • Citations for publications from Pubmed may now be searched and imported.
    • Delegated assistant access has expanded for users with the “ASST” role.
    • “NIH Support” sections have been relabeled to “NIH and Other Agency Sources of Support on Record.”
    • The column for “Funding Source” in Table 4 interprets the funding agency as NIH, AHRQ, or Other Fed.
    • Calculated means across all years have an increased precision, displayed with up to one decimal point.
    • Output PDF documents now have internal bookmarks linking to the beginning of each training table.

    Fixes

    • A defect in Table 8 generation that allowed duplicate citing of the same source within a training year is now corrected.
    • Navigating to the list of Participating Faculty for an RTD should now see reduced response times and increased performance.

    Post-release details and screenshots will be made available through Online Help for xTRACT resources. Review the original article in the attached PDF or on the eRA News website.

    Posted by Office of Sponsored Programs | 08/17/16
  • Comprehensive Cancer Center 18th Annual Research Retreat

    UAB Comprehensive Cancer Center

    18th Annual Research Retreat – Monday, October 17, 2016

    Sheraton Hotel Downtown Birmingham

    2016 Meeting Theme – Immunotherapeutics

    Program Chairs:  Ravi Bhatia, M.D. and Harry Erba, M.D., Ph.D.

    Meeting Website:  http://www3.ccc.uab.edu/index.php/research/research-retreat/  

    Abstract submission: August 1 – September 16, 2016

    Online Registration: August 1  – September 16, 2016

    RESEARCH COMPETITION

    Abstract submission: Monday, August 1  – Friday, September 16, 2016

    Eligible participants may submit an abstract of their original cancer research for presentation as a poster in the research competition.  First, second, third place and honorable mention awards are presented for the most outstanding posters.  Winners receive cash awards.  See meeting website for details.

    Cancer Research – Open Category (non-award)

    Cancer researchers not eligible for participation in an award category are encouraged to submit an abstract for presentation as a poster in the Cancer Research – Open Category.  This non-award category offers the opportunity for researchers to discuss their work with meeting participants.

    MEETING PRESENTATIONS

    On-line registration:  Monday, August 1 – Friday, September 16, 2016 

    The annual Research Retreat provides the opportunity to stimulate interdisciplinary research collaboration and development among participants.  This year’s event features presentations by experts in the field of Immunotherapeutics.

    We hope to see you there!


    Attachments:
    Posted by | 08/11/16
  • UAB Extramural Support Checklist Updated

    A new version of the UAB Extramural Support Checklist is now available. Revisions include:

    • Fee for Service is available as a Submission Type and F&A Base on numbers 3 and 27 respectively.
    • In accordance with the OSP Review Plan, options for either a complete or limited review are available at the top of the form.
    • A check box for CME/CPE was added above the Dean’s signature on the signature page of the form.

    Please take this opportunity to download the 8/10/2016 version of the UAB Extramural Support Checklist from the Office of Sponsored Programs Forms webpage.

    Posted by Office of Sponsored Programs | 08/10/16
  • 4th Annual UAB Center for Exercise Medicine Symposium

    The 4th annual UCEM Symposium is set for Thursday, Sept. 22, at the Hill University Center (Ballroom). Register today (it's free!); submit an abstract for the poster competition, and become a center member. Visit our website to do all three: http://www.uab.edu/medicine/exercise/center-for-exercise-medicine-annual-symposium

    UAB Speakers:

    David Brown, PT, PhD, SHP Department of Physical Therapy
    Dori Pekmezi, PhD, SOPH Department of Health Behavior
    Ceren Yarar-Fisher, PT, PhD, SOM Department of Physical Medicine & Rehabilitation
    Rob Motl, PhD, SHP Department of Physical Therapy

    External Guests and Speakers:

    P. Darrell Neufer, PhD
    Director, East Carolina Diabetes and Obesity Institute
    Professor, Departments of Physiology and Kinesiology
    East Carolina University

    Krista Vandenborne, PT, PhD
    Professor and Chair, Department of Physical Therapy
    University of Florida

    Scott Trappe, PhD
    Professor and Director, Human Performance Laboratory
    Ball State University

    Anne McTiernan, MD, PhD
    Professor, Department of Epidemiology, School of Public Health
    Member, Fred Hutchinson Cancer Research Center
    University of Washington

    Mike Roberts, PhD
    Assistant Professor, School of Kinesiology
    Auburn University

    Posted by Center for Exercise Medicine | 08/08/16
  • NIH Extramural Nexus Updates - July 2016

    The NIH Extramural Nexus Updates for July 2016 have been released. Stories highlighted this month include:

    • Criterion Scores and Funding Outcomes
    • Data on Model Organism Funding
    • Fellowship Application Tips
    • And more…
    Posted by Office of Sponsored Programs | 08/02/16
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