Tom, Sorry for the long delay in posting our mounting our procedure. Here
it is. Hope it was worth the wait.
Tissue Clearing Procedure for thick slices
This procedure is used for clearing thick (300-500µm) slices of rat brain
tissue which has been fixed with 4% paraformaldehyde and is stored in
buffer.
1. Transfer the tissue to a glass container.
2. Wash twice with distilled water for 5-10 minutes.
3. Wash with graded alcohols (65%, 70%, 80%, 85%, 90%, 95% and absolute)
for 5-10 minutes each.
4. Wash with xylene for 5-10 minutes.
5. Wash with Histoclear® for 5-10 minutes.
6. Mount the tissue in a depression well slide with Methyl Salicylate and
seal the cover slip with clear nail polish.
Some of our early publications may also be helpful.
Deitch, J.S., Smith, K.L., Swann, J.W. and Turner, J.N. Ultrastructural
Investigation of Neurons Identified and Localized Using the Confocal
Scanning Laser Microscope. J. Electron Micrsosc. Tech. 18, 82-90, 1991.
Turner, J.N., Szarowski, D.H., Smith, K.L., Marko, M., Leith, A., and
Swann, J.W. Confocal Microscopy and Three-dimensional Reconstruction of
Electrophysiologically Identified Neurons in Thick Brain Slices. J.
Electron Microsc. Tech. 18, 11-23, 1991.
Deitch, J.S., Smith, K.L., Swann, J.W., and Turner, J.N. Parameters
Affecting Imaging of the Horseradish Peroxidase-diaminobenzidine Reaction
Product in the Confocal Scanning Laser Microscope. J. Microsc. 160,
265-278, 1990.
Szarowski, D.H., Barnard, D.P., Smith, K.L., Swann, J.W., Holmes, K.V. and
Turner, J.N. Confocal Laser Scanned Microscopy: Analog Signal
Processing. Scanning 12, 265-272, l990.
Deitch, J.S., Smith, K.L., Chong, L.L., Swann, J.W. and Turner, J.N.
Confocal Scanning Laser Microscopic Images of Hippocampal Neurons
Intracellularly Labeled with Biocytin. J. Neurosci. Methods 33, 61-76,
1990.
Jim Turner
Tom Phillips wrote:
> Anybody willing to share their methyl salicylate clearing and subsequent
> coverslipping medium protocols? Thanks. Tom
>
> Thomas E. Phillips, Ph.D.
> Associate Professor of Biological Sciences
> Director, Molecular Cytology Core Facility
>
> 3 Tucker Hall
> Division of Biological Sciences
> University of Missouri
> Columbia, MO 65211-7400
> (573)-882-4712 (voice)
> (573)-882-0123 (fax)
|
