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Thin Sectioning of Slice Preparations for Immunohistochemistry

1,2, 2

1Department of Medicine, Yonsei University College of Medicine, Severance Hospital, 2Department of Psychiatry, Harvard Medical School

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    Summary

    The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

    Date Published: 4/28/2007, Issue 3; doi: 10.3791/194

    Cite this Article

    Park, J., Cunningham, M. G. Thin Sectioning of Slice Preparations for Immunohistochemistry. J. Vis. Exp. (3), e194, doi:10.3791/194 (2007).

    Abstract

    Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue. Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle. Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm. We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections. The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed. We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice. This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice. A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT). This is then quickly frozen with crushed dry ice and the resulting wafer can be position easily for cryostat sectioning Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

    Protocol

    1. Prepare mold from tape for OCT platform.
    2. Fill mold with OCT. Freeze within cryostat or by using crushed dry ice.
    3. Remove tape from around frozen OCT platform.
    4. Align marks on freezing chuck and cryostat mounting stage and lock in chuck.
    5. Section through OCT platform until surface is flat.
    6. Remove resurfaced OCT platform and place on cryostat freezing stage.
    7. Place tissue sample (previously cryopreserved with 30% glycerol or sucrose in PBS) in OCT.
    8. Prepare freezing column with outer ring projecting about 5 mm above top of column forming well for OCT.
    9. Carefully position tissue sample onto center of freezing column surface and slowly add OCT until well is filled.
    10. Surround freezing column with crushed dry ice. Tissue and OCT should completely freeze within 20-60 seconds.
    11. As preparation increases in temperature, the outer ring can be removed while the sample remains frozen.
    12. Slide sample off freezing column sideways and place in cryostat.
    13. Place drop of OCT on surface of OCT platform and position specimen (tissue down) applying firm pressure. Specimen will quickly freeze onto OCT platform.
    14. Secure chuck onto cryostat mounting stage with marks aligned.
    15. Section through OCT superficial to the tissue specimen.
    16. Thaw mount thin sections onto glass slides and store frozen or at room temperature.
    17. Immunoreactions can be performed for tissue mounted on glass slides.
    18. Reagent is pooled onto slide, can be gently agitated, and may be covered if light-sensitive.
    19. Subsequent stages of the reaction are easily performed by inverting slide into waste receptacle, wicking the slide, and then applying the next reagent.

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    Discussion

    The protocol presented here provides researchers with a concise, easy-to-follow outline of how to obtain thin cryostat sections of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices for further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry.

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    Disclosures

    Materials

    Name Company Catalog Number Comments
    O. C. T. Ted Pella, Inc. 27050
    Glycerol Solution 30% Glycerol Solution in PBS w/v
    Sucrose Solution 30% Sucrose Solution in PBS w/v

    References

    1. Scoutern, C. W., O'Connor, R., Cunningham, M. Perfusion fixation of research animals. Microsc. Today. 14, 26-33 (2006).
    2. Cunningham, M. G., Connor, C. M., Zhang, K., Benes, F. M. Diminished serotonergic innervation of adult medial prefrontal cortex after 6-OHDA lesions in the newborn rat. Brain Res. Dev. Brain Res. 157, 124-131 (2005).

    Comments

    3 Comments

    Thank-you for posting this video. Your device seems to work well with small tissue, but what do you use for freezing whole adult rat brains? Is the powdered dry ice method sufficient alone to reduce freezing artifact if you are freezing a whole adult brain? Do you have an alternative procedure for these applications?   Much appreciated,
    Neil
    Reply

    Posted by: AnonymousOctober 23, 2008, 4:04 AM

    Hi Neil, you actually can get a pretty good freeze with finely powdered dry ice. Just cover and let freeze for 3-5 minutes.  I would recommend, however, immersing the whole brain in isopentane that has been chilled (~30 minutes) in dry ice.  Just leave the brain in isopentane for 30 seconds, otherwise it can distort and fracture.  You will then need to let the (very cold, -80 degrees) brain equilibrate (warm up) to your cryostat or microtome temperature in order for it to cut smoothly.
    Good luck!
    Miles

    Miles G. Cunningham, MD PhD
    Director, Laboratory for Neural Reconstruction
    Administrative Director, McLean Fellowship in
    Neuropsychiatry and Behavioral Neurology
    Chair, ANPA Programs Committee
    Executive Director, Asniya, Inc.
    MRC 333, McLean Hospital
    Harvard Medical School
    115 Mill Street
    Belmont, MA  0²478
    office:     617.855.²051
    fax:         617.855.3199
    page:      617.855.²000
    Reply

    Posted by: AnonymousOctober 24, 2008, 5:18 PM

    Thanks for this very helpful video. I'm interested in knowing where you purchased the freezing rod and ring that is used to form the sample mold. Alternatively what metal is the rod made of and is there any significance to the length of the freezing rod used?
    Reply

    Posted by: Ellen Y.April 23, 2010, 3:57 PM

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