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the mouse is positioned along the edge of the table, with its forelimbs suspended
over the edge and allowed to move freely. Each forelimb (forelimb, second task;
hind limb, third task) is gently pulled down, and retrieval and placement are
checked. Finally, the mouse is placed toward the table edge to check for lateral
placement of the forelimb. The 3 tasks are scored in the following manner: normal
performance, 0 points; performance with a delay (2 sec) and/or incomplete, 1
point; no performance, 2 points. A total of 9 points means maximal neurological
deficit, and 0 points means normal performance. Additionally, the body weights
of all animals were checked weekly for 8 weeks.
Histology and immunohistochemistry
Histology and immunohistochemistry of brain sections were performed as de-
scribed previously [16], [17]. At the end of behavioral testing, each animal was
anesthetized and perfused through the heart with cold saline followed by 4%
paraformaldehyde in 0.1 M phosphate buffer. The brains were post-fixed in same
fixative for 24 hr, followed with followed with cryoprotection in 30% sucrose for
24 hr and then 30 µm sections were prepared on a cryostat (Leica CM 3000). The
sections through the needle entry site which was identifiable on the brain surface,
and sites 1.0 mm anterior and 1.0 mm posterior to plane were processed for X-gal
staining to analyze the hemisphere area. These sections are representative of the
core of the ICH lesion. The morphometric analyses involved computer-assisted
hand delineation of the area of the striatum, cerebral cortex, and ventricles, as
well as the whole hemisphere. Adjacent serial coronal sections were processed for
double immunofluorescence staining of human nuclear matrix antigen (hNuMA,
1:100, mouse monoclonal, Oncogene) and antibodies specific for cell type spe-
cific markers. Antibodies specific for neurofilament low molecular weight protein
(NF-L, 1:1000, rabbit, Chemicon), neurofilament high molecular weight protein
(NF-H, 1:1000, rabbit, Chemicon), microtubule associated protein-2 (MAP2,
1:500, rabbit, Chemicon), glial fibrillary acidic protein (GFAP, 1:1000, rabbit,
DAKO, Carpinteria, CA), and phospho-Akt1 (1:100, rabbit, Upstate) were used
for cell type identification of neurons and astrocytes. Brain sections were incu-
bated in mixed solution of primary antibodies overnight at 4°C as free floating
sections, followed by mixed secondary antibodies of Alexa Fluor 488-conjugated
anti-mouse IgG (1:1000, Molecular Probes, Eugene, OR) and Alexa Fluor 594-
conjugated anti-rabbit IgG (1:1000, Molecular Probes) for 1 hr at RT. Negative
control sections from each animal were prepared for immunohistochemical stain-
ing in an identical manner except the primary antibodies were omitted. Stained
sections were then examined under an Olympus confocal laser scanning biologi-
cal microscope (Olympus, Tokyo, Japan).
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