Biology Reference
In-Depth Information
slices or immunolabeled slices can be stored in the cold PBS.
The preparation of fi xed brain slices is the same as the spinal
cord.
3. Preparation of frozen slices
To prepare the cryosections, the frozen tissue is fi rst embedded
in OCT and then is sectioned into about 20
m or less sections
by a cryotome. The cryosections are picked up by gelatin-
coated slides and stored at −80°C.
4. Imaging of fresh tissues
The fresh spinal or brain tissue is placed into the imaging cham-
ber (Fig. 3a ). Here a cover glass-bottom imaging chamber is
essential to acquire a high-quality image. The cover glass-bot-
tom dish from MatTek Corporation is good for a short time
imaging. A long time imaging needs the sample to be circu-
lated with Krebs' solution so that the imaging chamber con-
nected with tubes for circulation from Harvard Apparatus is a
good choice. To prevent the sample from fl oating in the solu-
tion, a small piece of mesh is necessary to hold the sample at
the bottom of the chamber. The mesh can be attached on the
chamber by using vacuum grease so that the imaging chamber
can be reusable. The mesh with a holder is also commercially
available for the imaging and recording chamber. To keep the
sample's bioactivity, Krebs' solution fi lled in the imaging cham-
ber has to be oxygenated all the time during the imaging.
5. Imaging of fi xed samples
For imaging of a thick fi xed sample, the imaging chamber is
the same as that for the fresh tissue but without oxygen input.
For imaging of a fi xed slice with a thickness below 100
μ
m, it
is more convenient to use two pieces of cover glass forming a
μ
Fig. 3. Sample preparation for CARS imaging of myelin. ( a ) CARS imaging of a fresh tissue. The fresh tissue is placed in the
imaging chamber with cover glass bottom and covered by a piece of mesh. The imaging chamber is fi lled with oxygenated
Krebs' solution. ( b ) Schematic of CARS imaging of a fi xed slice. The fi xed slice surrounded by PBS is placed between two
cover glasses which are attached together by double sided tape.
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