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

  • Agency for Healthcare Research and Quality (AHRQ) Multiple Program Director/Principal Investigator (PD/PI) Policy

    The Agency for Healthcare Research and Quality have announced a policy affecting the acceptance of Multiple Program Director(s)/Principal Investigator(s) in appropriate research grant applications. This policy is effective with new applications submitted on or after October 1, 2016.

    Topics discussed within the policy announcement include:

    • Availability of Multiple-PD/PI Model
    • Change from an active single-PD/PI to a multiple-PD/PI award, or changes in the number or makeup of multiple-PD/PIs.
    • Change from an active multiple-PD/PI to a single-PD/PI award.
    • Deciding When to Use the Multiple-PD/PI Model

    Please review the original policy notice here (NOT-HS-16-018) for more details.

    Posted by | 09/26/16
  • NIH Notices - September 19, 2016

    Notices:
    • Policy on Funding Opportunity Announcement (FOA) for Clinical Trials
      (NOT-OD-16-147) National Institutes of Health
    • Policy on Good Clinical Practice Training for NIH Awardees Involved in NIH-funded Clinical Trials
      (NOT-OD-16-148) National Institutes of Health
    • NIH Policy on the Dissemination of NIH-Funded Clinical Trial Information
      (NOT-OD-16-149) National Institutes of Health
    See the full web version of these notices and more here.
    Posted by Office of Sponsored Programs | 09/19/16
  • Joint Symposium on HIV Research in Women

    The goal of the Inter-CFAR collaborative symposium on HIV in Women is to share cutting edge research, extend knowledge and foster action to address gaps toward understanding the complex clinical, biological and social aspects of HIV in women. This meeting will be held in conjunction with the 2016 Women’s Interagency HIV Study (WIHS) EC meeting to allow for joint activities with WIHS investigators. The meeting will consist of three sessions:
    • Vulnerable Populations
    • Microbiome in HIV-Infected Women and its Impact on Health Outcomes
    • The HIV Continuum of Care Across the Lifespan of Women
    We are currently soliciting abstracts for posters and oral presentations from investigators in relevant areas until Oct 2, 2016.
    Posted by Center for AIDS Research | 09/15/16
  • Research Administration Announces Melinda Cotten as Assistant Vice President for Sponsored Programs

    ​Dr. Richard Marchase, Vice President for Research and Economic Development, is pleased to announce the appointment of Melinda Cotten as Assistant Vice President of Sponsored Programs. She will be starting on September 19, 2016. Ms. Cotten will be working with her staff in assisting UAB researchers in their efforts to secure and fulfill extramurally funded sponsored programs by performing pre-award and non-financial post-award administration. With a focus on customer-centric approaches for continuously improving UAB’s processing of submissions, Cotten’s extensive experience will be critical to ensuring the continued success of the UAB research community, and maintaining UAB’s national position as a top research university.
    Posted by Office of the Vice President for Research and Economic Development | 09/12/16
  • NIH Notices – September 12, 2016

    Notices

    • Optional Electronic Method to Request Withdrawal of Applications from Consideration for Funding
      (NOT-OD-16-143) National Institutes of Health
    • Notice of Correction to PA-16-309 "Ruth L. Kirschstein National Research Service Award (NRSA) Individual Predoctoral Fellowship (Parent F31)"
      (NOT-OD-16-146) National Institutes of Health
    • AHRQ Retires Policy on Post-Submission
      (NOT-HS-16-017) Agency for Healthcare Research and Quality
    Posted by Office of Sponsored Programs | 09/12/16
  • National Science Foundation (NSF) System Shutdown on September 17-18, 2016

    All National Science Foundation (NSF) systems, including the NSF websiteResearch.gov and FastLane, will be unavailable from 5:00 PM on Saturday, September 17, until 4:00 PM on Sunday, September 18. This downtime is scheduled to coincide with repairs to the Foundation's electrical systems. Affected deadlines that fall on Monday, September 19, are extended to Tuesday, September 20, submitter's local time. See the attached NSF downtime notice for more information on affected solicitations.

    Attachments:
    Posted by Office of Sponsored Programs | 09/09/16
  • eRA Enhancements – September 9, 2016

    Effective September 8, 2016, Signing Officials have the ability to electronically request the withdrawal of an application through eRA Commons. A Signing Official has two business days after successfully submitting an error-free application to reject the application. If more than two business days have elapsed, the Signing Official must contact the Division of Receipt and Referral (DRR) to request an application be withdrawn. Contacting DRR directly may now be done electronically. Requesting the withdrawal of an application will remain available while the application has not undergone review and the summary statement posted.

     

    Features

    • The initiation of a withdrawal request can be done by the Signing Official or Principal Investigator.
    • A new navigation tab, Prior Approval, was added to Commons.
    • Signing Officials have the ability to search for requests.
    • Automated notifications are generated on submission and on approval of DRR withdrawal requests.

     

    For more details on the features listed above, please review the original announcement. For more information concerning Prior Approval for Withdrawals, please see Guide Notice NOT-OD-16-143 and the Prior Approval section in the eRA Commons online help.

    Posted by | 09/09/16
  • National Heart, Lung, and Blood Institute (NHLBI) Years of Support Clarification for K24 Funding

    The National Heart, Lung, and Blood Institute (NHLBI) have announced an important clarification impacting years of support for both new and renewal K24 funding. The maximum years of support is capped at 10 years. Review the full notice for more details.

    Posted by Office of Sponsored Programs | 09/08/16
  • NIH Extramural Nexus Updates - August 2016

    The NIH Extramural Nexus Updates for August 2016 are now available. Some highlighted stories include:

    • NIH Support for Model Organisms, Parts Two and Three
    • National Research Service Awards (NRSAs) Postdoctoral Stipend Guidelines for Fiscal Year 2017
    • NIH Loan Payment Programs (LRPs) Accepting Applications from September 1 through November 15
    • Changes to Post-submission Materials and Appendix Policies Take Effect January 25, 2017
    • Withdrawing Applications Electronically in eRA Commons, Available on September 8, 2016
    • FAQ: How May a PI Check Their Eligibility for a Continuous Submission?
    • NIH Closed for Federal Holiday: September 5, 2016 (Labor Day)
    • See the original digest or attachment for more information.
    Posted by Office of Sponsored Programs | 09/02/16
  • New F&A Rate - Implementation for Proposals in Process

    UAB has a new Facilities and Administrative (F&A) Rate Agreement with the federal government.  Effective immediately, all Principal Investigators and department staff should update all in-process and future proposals, particularly for the NIH Oct. 5th deadline to the new rates.  The On-Campus rate for research is 48.5% and the Off-Campus (more than 50% of the project is off campus and rent is included in the budget) rate remains the same at 26%.  All fringe benefit rates remain the same.
     
    This rate agreement is dated 9/1/2016 and is effective until 9/30/2019.
     
    If you have any questions or concerns, please contact the Office of Sponsored Programs by email: askosp@uab.edu.
    Posted by | 09/01/16
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