Immunological Response to Experimental Intracerebral Hemorrhage: Morphological Assessments - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 606

2. Cut the midline of animal's chest with scissors.
3. Cut through the diaphragm and lift sternum with forceps to
expose the beating heart.
4. Insert the perfusion cannula, connected to a perfusion pump,
into the left ventricle. Make an incision into the right atrium to
allow blood to fl ow out of the vascular system.
5. Perfuse animal with ice-cold PBS 0.1 M at pH 7.4 until the
draining perfusion solution becomes clear.
6. Then switch to 4% PFA. The total perfusion volume is approxi-
mately 1 ml/1 g body weight (about half and half for PBS and
PFA solutions).
7. The perfusion rate should not exceed 20 ml/min to avoid
damage to the vasculature. The perfusion rate in our protocol
is 15 ml/min.
Note : An alternative to PBS is normal saline or Millonig's
Buffer.

1. Brain tissue from the perfused animals should then be gently
removed from the cranial cavity using appropriate dissection
tools.
2. The excised brain tissue should be post-fi xed overnight in the
same fi xative solution (4% PFA in 0.1 M PBS) on 3D rotator
at room temperature.
3. On the next day rinse brain three times, 10 min each on the
rotator in freshly prepared solution 0.1 M PBS at pH 7.4 to
remove PFA.
4. Store post-fi xed brains in PBS 0.1 M at pH 7.4 at 4°C until
usage.

2.3. Post-fi xation

Note : Avoid over-fi xation as it affects antigen accessibility.
There are several options for storing tissue for later processing.
Perfused tissue, fi xed in 4% PFA overnight can be stored in PBS at
4°C for months. Also, tissue can be cryoprotected by incubation
with 30% sucrose until the tissue sinks (see below), then embedded
in Optimal Cutting Temperature (OCT) compound and saved at
−80°C until further tissue processing. For free-fl oating immunos-
taining, 30- m (or thicker) tissue sections can be saved in cryopro-

tectant at 4°C for several years.
Only the protocol for free-fl oating brain sections is discussed
below.

μ

Materials and solutions
1. Cryostat (Leica CM1850, Leica Microsystems GmbH, Wetzlar,
Germany).
2. 3D rotator (Fig. 2 , #18).
3. Glass dish.

2.4. Brain Sectioning