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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.
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).
- Prepare mold from tape for OCT platform.
- Fill mold with OCT. Freeze within cryostat or by using crushed dry ice.
- Remove tape from around frozen OCT platform.
- Align marks on freezing chuck and cryostat mounting stage and lock in chuck.
- Section through OCT platform until surface is flat.
- Remove resurfaced OCT platform and place on cryostat freezing stage.
- Place tissue sample (previously cryopreserved with 30% glycerol or sucrose in PBS) in OCT.
- Prepare freezing column with outer ring projecting about 5 mm above top of column forming well for OCT.
- Carefully position tissue sample onto center of freezing column surface and slowly add OCT until well is filled.
- Surround freezing column with crushed dry ice. Tissue and OCT should completely freeze within 20-60 seconds.
- As preparation increases in temperature, the outer ring can be removed while the sample remains frozen.
- Slide sample off freezing column sideways and place in cryostat.
- Place drop of OCT on surface of OCT platform and position specimen (tissue down) applying firm pressure. Specimen will quickly freeze onto OCT platform.
- Secure chuck onto cryostat mounting stage with marks aligned.
- Section through OCT superficial to the tissue specimen.
- Thaw mount thin sections onto glass slides and store frozen or at room temperature.
- Immunoreactions can be performed for tissue mounted on glass slides.
- Reagent is pooled onto slide, can be gently agitated, and may be covered if light-sensitive.
- 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.
|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|
- Scoutern, C. W., O'Connor, R., Cunningham, M. Perfusion fixation of research animals. Microsc. Today. 14, 26-33 (2006).
- 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).
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