Assessments of Reactive Astrogliosis Following CNS Injuries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 60

3. Carefully remove brain and/or spinal cord for post-fi xation
using dissection tools.
4. Place extracted tissue in 4% phosphate-buffered PFA for post-
fi xation for a minimum of 4 h to a maximum of 24 h at 4°C.
Brain: 40 ml (rat) or 10 ml (mouse) in individually labeled

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conical tubes.
Spinal cord: splinted in 40 ml (rat) or 10 ml (mouse) indi-

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vidually labeled conical tubes.
To prevent permanent curvature of the spinal cord -

after perfusion fi xation, the tissue needs to be kept in a
straight confi guration for at least 24 h.
Using a suitable splint (i.e., wooden applicator stick, -

tongue depressor) affi x the ends of the spinal cord to
render the normal curvature straight without pulling
or stretching the tissue. Spinal cord tissue can be
affi xed with suture or adhesive (i.e., cynoacrylic). Note
that the ends used for tethering will be damaged and
should not contain areas of experimental interest.
5. Cryoprotect (when appropriate) by sucrose-imbedding via
incubation (4°C) in increasing gradients of sucrose (10-30%)
for 24-48 h. See Note 6.
6. If using vibratome for sectioning, store tissue in PBS (4°C)
and omit cryoprotection step (above).

1. Cryostat sectioning:
Sucrose-infi ltrated fi xed tissue should be embedded in

3.2. Serial Tissue
Sectioning ●

OCT (Tissue Tek) in small mold in an orientation perpen-
dicular to plane of analysis.
Snap-freeze embedded tissue in isopentane over dry ice or

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in liquid nitrogen.
Affi x tissue block to cryostat (or microtome) chuck using a

●

small amount of OCT.
Stabilize the sample to cryostat temperature (typically

●

−20°C) before cutting.
Set the micrometer screw to the desired section thickness.

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- If stereological analysis is to be conducted to quantify

labeled cells, 30-50 mm sections should be obtained.
Thinner sections (i.e., 10-20 mm) can be obtained if
other quantifi cation methods will be used. Ultrathin
(i.e., >10 mm) sectioning is not generally necessary.
Tissue can be collected in 0.1 M PB in a collection -

plate (i.e., 24-well plate) or directly onto warm (40°C)
1% gelatin-subbed slides.