Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 607

4. Paint brush (Fig. 2 , #5).
5. 1.5-ml microcentrifuge tubes and storage box for tubes.
6. 0.1 M PBS, pH 7.4.
7. Cryoprotectant (see a recipe below).
8. Optimal Cutting Temperature (OCT) compound.
9. Dry ice.
10. Metal (lead) frame and pad.
Protocol
1. Transfer brain into 30% sucrose until brain sinks (~24 h) at
room temperature for cryoprotection.
2. Repeat rinses of brain tissue: three times, 10 min each on the
rotator in 0.1 M PBS solution.
3. Embed brain into OCT compound. Using a metal frame placed
on the metal pad (~0.5 cm thickness) place a small amount of
OCT. Then position brain on the OCT layer and place a metal
pad on dry ice for a few minutes to harden the OCT. Then,
complete brain coverage with an adequate amount of OCT.
4. Place metal pad on the dry ice until OCT is frozen (~15 min).
Note : avoid rapid tissue freezing as brain can fracture.
5. Open the metal frame and remove the frozen OCT cube hold-
ing the embedded brain.
6. Using OCT, attach the frozen OCT cube containing the brain
tissue to cryostat chuck and cut brain into 30- μ m section (or

other desired thickness) in the cryostat.
7. Place each 30- m section into a small dish containing 0.1 M

PBS to dissolve OCT and rinse the brain sections.
8. Using a paint brush, carefully transfer each 30-

μ

m brain sec-
tion into individual 1.5-ml microcentrifuge tubes fi lled with
1 ml cryoprotectant solution, label and store at 4°C. (This will
allow for long-term storage).
9. Mount each 10th section for standard Cresyl Violet (CV)
staining on Superfrost Plus slides.
Note : As option, a three-step cryoprotection can be performed
with gradual increase of sucrose concentration (10% to 20% to
30%). Each step requires waiting until brain sinks. This approach
allows better penetration of sucrose and reduces non-specifi c stain-
ing backgrounds.

μ

2.5. Double
Immunohisto-
chemistry Procedure
for Free-Floating
Brain Sections (Fig. 3 )

Materials, solutions, and reagents

1. 24-well plates. Multiple Well Cluster Plate, 24 Well, Tissue
Culture-Treated, Corning Costar #3526 (Fig. 2 ).

2. Paint brush.