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

  • July 2016 Issue of Compliance 411 Newsletter Published

    The University Compliance Office announces publication of the July 2016 issue of their quarterly newsletter, Compliance 411

    Among the topics in this issue are Responsible Conduct of Research at UAB; new Recertification of Effort Form and process; a profile of Research Integrity Officer Pam Bounelis, PhD; defining fabrication, falsification and plagiarism; new research data management requirements; and the new chemical inventory system.

    Your comments and feedback regarding Compliance 411 are welcome; please contact compliance@uab.edu.

    Attachments:
    Posted by University Compliance Office | 07/22/16
  • UAB Industry-Sponsored Clinical Trials F&A Rate Will Increase Effective July 1, 2016

    Effective July 1, 2016, the UAB Facilities & Administrative (F&A) rate for industry sponsored or funded clinical trials will increase to 30%. This change is only applicable to new submissions to the Office of Sponsored Programs (OSP) on or after July 1, 2016.
    Posted by Office of Sponsored Programs | 07/01/16
  • Recertification of Effort Form and Process Change

    Effective July 1, 2016, the Recertification of Effort Form will be used for recertifying a previously certified effort report and certifying a delinquent effort report. The Recertification of Effort Form, which replaces the form entitled ORC Review Request (commonly referred to as a "request to recertify effort"/"RFR"), is listed as follows on the Financial Affairs forms site:

    OSP-FA-Effort-160701.jpg

    The new form was drafted with considerable feedback from stakeholders on campus.  Enhancements to the form include:

    • More specific questions to be addressed by the submitter in order to help mitigate follow-up and expedite the review and approval;
    • Clear roles and responsibilities for each signing official; and
    • An accompanying informational attachment of the effort recertification process.

    In addition, there is a routing change. The effort recertification forms are to be forwarded directly to Financial Affairs by the Dean’s offices upon completion and approval rather than to the University Compliance Office (UCO). If you have any questions regarding this new process change, please contact the University Compliance Office at (205) 996-6540.

    Posted by Office of Sponsored Programs | 07/01/16
  • Search Opened for Vice President of Research

    The UAB Office of Human Resources has posted information on the search for the next Vice President of Research, including information about the Opportunity, Search Committee membership, and current stage of the search process. 
    Posted by Office of the Vice President for Research and Economic Development | 07/01/16
  • UAB Research-Related Fee-for-Service (FFS) change effective July 1, 2016

    Currently, Fee for Service (FFS) agreements are processed at UAB by University Contracts and are assessed a 5% of revenue administrative processing fee. As Facilities and Administrative (F&A) costs are not currently assessed on those FFS activities related to research, UAB academic units do not receive any F&A distributions in support of their research missions, despite the fact that these FFS activities result in true research infrastructure expenses. In addition, current FFS activities are not counted toward UAB’s national metrics on research expenditures (e.g., NSF HERD, etc).

    As such, and consistent with practices of peer institutions, all agreements (and contract renewals) with terms beginning on or after July 1, 2016 for research-related FFS contracts will be processed by the Office of Sponsored Programs (OSP) and be assessed F&A at a rate of 30% on cash received. Budgets for research-related FFS contracts should include recovery of full direct costs in accordance with the University’s Direct Costs Policy (available on Financial Affairs website) and the appropriate F&A rate as described above.

    Posted by Office of Sponsored Programs | 06/06/16
  • OSP Announces Streamline of NCC Submission Process and Updated Checklists

    ​In a continuing effort to streamline submission processes, the Office of Sponsored Programs is pleased to announce that NIH Research Performance Progress Report submissions (also known as RPPRs), other Noncompeting Continuation/Continuation submissions, and Pre-Application submissions no longer require a signed UAB Extramural Support Checklist, but should now be sent to osp@uab.edu with a UAB Expedited Checklist.
    Posted by Office of Sponsored Programs | 05/27/16
  • NIH Proposal Submission Revised Procedure

    The UAB Office of Sponsored Programs is pleased to announce the newly revised procedure for review of NIH proposal submissions for large submission deadlines involving NIH Research Project Grants (R01), Research Career Development Awards (K) and Research Fellowship (F) proposals. In order to review NIH proposals as quickly as possible, OSP has implemented a proposal review pool which will allow OSP to introduce additional reviewers to assist with proposal review when needed.

    NIH proposal submissions to OSP for deadline dates of June 5th and July 5th (R01), June 12th and July 12th (K) and August 8th and 13th (F), will  be assigned rotating through a  pool of reviewers instead of an assignment to a specific officer by department. The reviewer will be identified in the Receipt of Submission email once a complete submission is received by OSP. This change is effective immediately.

    NOTE: This is only applicable to new NIH proposal submissions for the above mentioned NIH deadlines. All other NIH proposal submission deadlines will be routed to the officer assigned to your specific department. All NIH awards will continue to be processed by the OSP officer assigned to your specific area.

    Please continue to follow the OSP Review Plan to ensure that your reviewer has sufficient time to properly review the proposal. 

    Please forward any questions regarding the NIH proposal review process to Tim Parker at tmparker@uab.edu or askosp@uab.edu.

    Posted by Office of Sponsored Programs | 05/26/16
  • New Guidance Information on IRB Website

    The OIRB has provided updated guidance information on our website.  New information has been posted regarding the following:
    • Flowchart to determine who are key personnel
    • NIH description of ethnic and racial categories
    • IRB form compatibility

    Key Personnel

    The IRB has created a flow chart to help determine whether individuals involved with research should be considered to be key personnel involved in the conduct of human subjects research.  The Key Personnel Flowchart is available under the General category on the Other Guidance page of the IRB website.

    Ethnic and Racial Categories

    The table on the Investigator's Progress Report follows the reporting requirements of the NIH. The following is their description of how the ethnic and racial categories are defined:​​

    Ethnic Categories

    • ​​Hispanic or Latino: A person of Cuban, Mexican, Puerto Rican, South or Central American, or other Spanish culture or origin, regardless of race.  The term “Spanish origin” can also be used in addition to “Hispanic or Latino.
    • Not Hispanic or Latino​.

    ​​​​​​Racial Categories

    • ​American Indian or Alaska Native: A person having origins in any of the original peoples of North, Central, or South America, and who maintains tribal affiliations or community attachment.​
    • Asian: A person having origins in any of the original peoples of the Far East, Southeast Asia, or the Indian subcontinent including, for example, Cambodia, China, India, Japan, Korea, Malaysia, Pakistan, the Philippine Islands, Thailand, and Vietnam. (Note: Individuals from the Philippine Islands have been recorded as Pacific Islanders in previous data collection strategies.) 
    • Native Hawaiian or Other Pacific Islander: A person having origins in any of the original peoples of Hawaii, Guam, Samoa, or other Pacific Islands.
    • Black or African American: A person having origins in any of the black racial groups of Africa. Terms such as “Haitian” or “Negro” can be used in addition to “Black or African American.”
    • White: A person having origins in any of the original peoples of Europe, the Middle East, or North Africa.

    IRB Form Compatibility Issues

    IRB forms on this page are compatible with Microsoft Word 2010 and newer on Windows and Microsoft Word 2016 for Mac. Some features (e.g., checkboxes) are not compatible with older versions of Microsoft Word or any version of Pages for Mac. The newest versions of Microsoft Word are available at no charge to UAB faculty, staff, and students through UAB IT. Contact UAB IT for more information on upgrading your software. If you are unable to upgrade to a compatible version of MS Word, you may simply place an "X" before the checkbox that corresponds to your answer in the form.
     
    Posted by Office of the Institutional Review Board for Human Use | 04/22/16
  • 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
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