Biology Reference
In-Depth Information
stereological cell counts
Total number of human NuMA-positive F3 (n = 3) and F3.Akt1 (n = 3) hNSCs
in the brain sections from ICH animals was determined by stereological estima-
tion as described previously [16], [17]. The sections used for cell count covered
the entire striatum with hemorrhage lesion and overlying cortex. This generally
yielded six or seven sections in a series. Sampling was done using the Computer
assisted stereological toolbox system, version 2.1.4 (Olympus), using an Olym-
pus BX51 microscope, a motorized microscope stage (Prior Scientific, Rockland,
NY) run by an IBM compatible computer, and a microcator (Heidenhain ND
281B, Schaumberg, IL) connected to the stage and feeding the computer with the
distance information in the z-axis. The counting areas were delineated at a 1.25×
objective and generated counting areas of 150×150 µm. A counting frame (1612
µm2) was placed randomly on the first counting area and systemically moved
through all counting areas until the entire delineated area was sampled. Actual
counting was performed using a 100× oil objective. Guard volumes (4 µm from
the top and 4-6 µm from the bottom of the section) were excluded from both
surfaces to avoid the problem of lost caps, and only the profiles that came into
focus within the counting volume (with a depth of 10 µm) were counted. The
estimate of the total number of HuNuMA-positive F3 and F3.Akt1 calculated
according to the optical fractionator's formula [35].
statistical Analysis
The statistical significance between group comparisons for behavioral data was
determined by one-way ANOVA and two-way ANOVA. P values<0.001 were
considered to be statistically significant (version 12.0, SPSS, Chicago, IL).
results
Stable Human Neural Stem Cell Line Overexpressing Akt1
F3 hNSC line was infected with a retroviral vector encoding mouse Akt1 gene
(Figure 1A), and clones resistant to hygromycin were selected and expanded. One
of the clones was chosen and used in the present study. The morphology of the
selected hNSC line, F3.Akt1 does not differ from the parental F3 hNSCs with bi-
polar- or multipolar-morphology (Figures 1B, C). Results of RT-PCR analysis of
mRNAs isolated from F3 and F3.Akt1 cells are shown in Figure 1D. Transcripts
for nestin (an NSC specific marker), neurofilament triplet proteins (NF-L, NF-M
and NF-H, cell type-specific markers for neurons), glial fibrillary acidic protein
Search WWH ::
Custom Search