Imaging of Myelin by Coherent Anti-Stokes Raman Scattering Microscopy - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 273

(b) Cold phosphate buffer solution (PBS); 0.1 M, pH = 7.2;
(c) Fixative solution: 4% paraformaldehyde in PBS solution;
(d) Oxygen tank with the tubes to supply the fresh tissue with
oxygen;
(e) Imaging chamber with bottom being cover glass (MatTek
Corporation, Ashland, MA or Harvard Apparatus Inc.
Holliston, MA);
(f) Cover glass;
(g) Dow Corning ® high vacuum grease;
(h) Nylon mesh (Walmart);
(i) Double sided tape;
(j) Gelatin-coated slides.

Fresh tissues are able to be used for the study of dynamics
of myelin changes within various controlled environments. Fixed
tissues or slices are good for the examination of morphology of
myelin at certain stages. The fi xed slices are also used for the com-
bination of CARS imaging of myelin and immuno-TPEF imaging
(TPEF imaging of immunolabels) of other proteins. Cryosections
are good for CARS imaging of some specifi c frozen tissues, such
as tissues from MS patients. During CARS imaging process, all
the samples must be placed in a wet environment and cannot be
in a dried or dehydrated state.
1. Preparation of fresh spinal cord and brain samples
After the animal is deeply anesthetized, a transcardial perfusion
is performed with the cold PBS (pH = 7.2). The spine is
extracted immediately from the animal followed by extracting
the spinal cord from the vertebrae carefully. Then, the spinal
cord is fi rst split into two halves by saggital division and then
cut radially to separate the ventral column from dorsal column.
The isolated ventral or dorsal strip is kept in oxygenated Krebs'
solution for an hour for recovery.
To image myelin in the fresh brain tissue, the extracted
brain is sectioned into at least 400-500 m brain slices by a

vibratome. The brain slices are kept in the oxygenated Krebs'
solution for an hour for recovery prior to imaging.
2. Preparation of fi xed samples
To better keep the structure of myelin, a transcardial perfusion
is performed with the cold PBS followed by cold fi xative solu-
tion after deep anesthetization of the animal. The spine with
vertebrae is extracted and kept in the fi xative solution for at
least 2 h. Then, the spinal cord was extracted from the spine and
stored in the fi xative solution. The fi xed spinal tissue is sec-
tioned into a certain thickness depending on the need for only
CARS imaging about 20-200

μ

μ m or for combining immuno-

TPEF imaging about 20-80 μ m by a vibratome. The fi xed