Thin Sectioning of Slice Preparations for Immunohistochemistry

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.

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 surf....

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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Materials

List of materials used in this article

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