Biomedical Engineering Reference
In-Depth Information
Note
*1: Information on observation time and day, animal's developmental stage (i.e.,
post-birth day), housing condition, and experimental environments (new or
familiar cage/room, or which fi eld location) is also critical.
*2: This crushed dry ice powder and labeling of the plastic embedding mold
should be prepared before collecting the animal brain. Do not let the animal
hear extraneous noise, as this will cause IEG induction in auditory pathways.
After behavior or sensory stimuli have been completed, then within the next
5-10 min, the animals should be sacrifi ced and the brain collected to avoid
RNA degradation and handling time effect. Cooling speed should be quick-
ened by adding ethanol as ethanol-dry ice bath.
9.3
Brain Sectioning and Stock
1. Put the OCT frozen block on sectioning stage in a cryostat and equilibrate to
−20 °C for 30 min (Fig.
9.1a
).
2. Trim excess OCT by a razor blade (Fig.
9.1b
).
3. Slice the sample into 10-12
m {
Note *1
} thick sections (Fig.
9.1c
). For brain
and embryonic tissue, the best cutting temperature is within the range of −18 to
−20 °C {
Note *2
}.
4. Mount frozen sections on the face of Superfrost Plus Microscope Slides [Fisher,
cat#12-550-15] {
Note *3
} (Fig.
9.1d-e
).
μ
Note
*1: Optimal section thickness for obtaining good-quality sections depends on
tissue types, age, and animal species. We recommend performing test sec-
tioning and Nissl staining.
*2: It is critical to adjust the cryostat temperature for the appropriate tissue type.
The position of the antiroll plate should allow space between the knife and
glass plate and also not touch the block when cutting. A thin, fi rm brush
allows unfolding and other manipulation of sections.
*3: Glass slides must be chemically coated, such as with silane, to reduce occur-
rence of detachment of tissue slices during procedures.
Fig. 9.1
Brain sectioning in a cryostat. (
a
) Setting of OCT frozen block on sectioning stage.
(
b
) Trimmed OCT frozen block. (
c
) Slicing of the sample. (
d
) Unfolding of sliced samples using
a fi ne brush. (
e
) Mounting of frozen section on the slide
