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Thin Sectioning of Slice Preparations for Immunohistochemistry
Jae-Joon Park1, 2, Miles G. Cunningham2
1Department of Medicine, Yonsei University College of Medicine, Severance Hospital, 2Department of Psychiatry, McLean Hospital, Harvard Medical School

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0:05 Introduction5

0:21 Subscription Lock21

0:30 Mold preparation30

4:40 Sectioning280

5:47 Immunoreaction347

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.

 

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  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.

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.

Name  Type  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

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)

Park JJ, Cunningham MG.. (2007). Thin Sectioning of Slice Preparations for Immunohistochemistry. JoVE. 3. http://www.jove.com/index/details.stp?id=194, doi: 10.3791/194
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10/23/2008 4:04:09 AM
Neil M. Fournier responded with a statement of type: Neutral

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

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10/24/2008 5:18:10 PM
MilesC responded with a statement of type: Neutral

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

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04/23/2010 3:57:04 PM
ellenyangster responded with a statement of type: Neutral
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?
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04/28/2007

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doi: 10.3791/194 

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Journal of Visualized Experiments (JoVE) is an online research journal employing visualization to increase reproducibility and transparency in biological sciences.

 

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