Assessment of Microthromboembolism After Subarachnoid Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 371

3. Procedures

3.1. Preparation for
Immunohistochemical
Staining

Have all solutions available.

3.2. Brain Sectioning

Sacrifi ce the animal at a given time point after induction of SAH.
Perfuse the animal through the left cardiac ventricle with NaCl,
0.9%, 50 ml, followed by 4% paraformaldehyde in phosphate-
buffered saline, 120 ml, over 20 min.
Remove the brain from the skull and fi x in 4% formaldehyde for
48 h.
Place brain into the matrix, ventral side up.
Line up brain longitudinally with matrix.
Slice brain with wet blades at intervals of 1 mm in mice (generally
6-7 slices).
Lift brain slices and blades together off the matrix.
Embed the slices in paraffi n using standard histology techniques.
Cut 7 μ m sections using a microtome (Leica, Wetzlar, Germany).

3.3. Immunohisto-
chemical Staining

Incubate the paraffi nized sections in xylene and rehydrate through
a decreasing gradient of ethanol solutions for deparaffi nization.
Retrieve antigen by heating the sections for 25 min in 0.01 mM
sodium citrate (pH 6.0) at 96°C.
Quench endogenous peroxidase activity by incubating the sections for
30 min in 0.3% H 2 O 2 in water.
Block the sections with 10% normal goat blocking serum in phos-
phate-buffered saline for 20 min.
Incubate with fi brinogen fi rst antibody (concentration depends on
antibody and experimentation) for 60 min.
Wash the sections with phosphate-buffered saline and incubate
with secondary biotinylated anti-body for 30 min.
Visualize antibody staining using antibody detection kit, such as
Vectastain ABC method according to manufacturer's instruc-
tions (Vector).
Counterstain with 0.5% methyl green.

3.4. Measuring
Microthrom-
boembolism

This can be done qualitatively by simple visual assessment of positive
staining (Fig. 1 ). More quantitative methods could be to grade the
degree of microthromboembolism on a 3-point semiquantitative
scale (none, some, a lot) and validate this by repeated, blinded
assessment of sections by two observers with calculation of inter
and intraobserver variability. Finally, quantitative methods can be