Acute Physiologic and Morphologic Assessment Following Subarachnoid Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 294

Fig. 3. MCAO induces reactive gliosis and nuclear translocation of STAT3 in astrocytes. At 24 h following MCAO in young
rats, enhanced immunostaining of GFAP-positive cells (astrocytes) was observed in ipsilateral cortex ( b ) compared to the
GFAP immunostaining observed in contralateral cortex ( a ). Basal GFAP immunostaining was observed in contralateral hip-
pocampus ( c ) that was enhanced in ipsilateral hippocampus ( d ). MCAO also resulted in the appearance of STAT3 immuno-
reactivity in astrocytic nuclei ( b , d , f ). ( b ) Western blot analysis of STAT3 activation following MCAO in rodents. At 6, 24, and
72 h following MCAO, STAT3, and pSTAT3 expression were evaluated quantitatively by immunoblot analysis. (DiNapoli et al.
2010).

Total RNA is isolated using Trizol ® reagent (Invitrogen; Carlsbad,
CA). Concentration and purity of RNA was determined using a
biophotometer and considered for use only if A260/A280 was
between 1.8 and 2.1. Total RNA (1

2.5. RNA Isolation,
cDNA Synthesis,
and Real-Time PCR
Amplifi cation

g) is reverse-transcribed to
cDNA using SuperScriptä III RNase H- and oligo (dT) 12-18
primers (Invitrogen) in a 40

μ

l reaction. Real-time PCR analyses
are performed using a PCR system (Applied Biosystems; Foster
City, CA) in combination with TaqMan ® chemistry. Glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) can be used as an endog-
enous control to normalize for differences in the amount of cDNA
added to reactions. Specifi c primers and dual-labeled internal fl uo-
rogenic (FAM/TAMRA) probe sets (TaqMan ® Gene Expression
Assays) for genes of interest are used according to the manufacturer's
recommendation (Applied Biosystems). All PCR amplifi cations
(40 cycles) are performed in a total volume of 50

μ

μ l, containing 1 μ l

cDNA, 2.5 μ l of the specifi c assay on Demand ® primer/probe mix,

and 25 l of TaqMan ® Universal master mix (Applied Biosystems).

Relative quantifi cation of gene expression is performed using the
comparative threshold (CT) method as described by manufacturer
(User Bulletin 2; Applied Biosystems). Changes in mRNA expres-
sion level were calculated following normalization to GAPDH and
expressed as fold change over corresponding controls.

μ