The Mechanism of Taurine Protection Against Endoplasmic Reticulum Stress in an Animal Stroke Model of Cerebral Artery Occlusion and Stroke-Related Conditions in Primary Neuronal Cell Culture - Advances in Experimental Medicine and Biology - page 230

23.2.2.3 Determination of Infarct Volume

Animals were sacrificed by isoflurane (Phoenix), and brains were removed for
2,3,5-triphenyltetrazolium chloride (TTC) staining and collecting samples for
Western blot (Kramer et al. 2010 ). Using an adult rat brain slicer (Matrix, Zivic
Instruments), brains were sectioned coronally into six 2 mm coronal slices
(2, 4, 6, 8, 10, and 12 mm from the frontal pole) and incubated for 5 min in a
2% (wt/vol) solution of TTC (J.T. Baker, India) at 37°C. TTC, a water soluble
salt, is reduced by mitochondrial dehydrogenases to formazan, which turns
normal tissue deep red (Bederson et al. 1986 ; Rich et al. 2001 ) . Thus, reduced
TTC staining identifies regions of diminished mitochondrial function in the
ischemic tissue. To assess lesion volume, TTC-stained slices were scanned
using an HP ScanJet 5530 and analyzed by Image J analysis software (public
domain software developed at NIH ( http://rsbweb.nih.gov/ij/ )). Lesion volume
was determined as the percent of the total ipsilateral hemispheric volume as
described previously (Swanson et al. 1990 ; O'Donnell et al. 2006 ) . Brie fl y, to
eliminate the effect of brain edema, the corrected infarct volume was calcu-
lated as follows: [(V R − V Ln )/V R ]100 in which V R is the volume of right hemi-
sphere and V Ln is the volume of nonlesioned tissue in left hemisphere (Schäbitz
et al. 1999, 2000 ; O'Donnell et al. 2006 ). After the TTC experiment, while the
sections were on ice, the ischemic parts of the left hemisphere (core and pen-
umbra) and the right hemisphere (identical regions) were quickly dissected
(Fig. 23.1a ) (Ashwal et al. 1998 ) .

23.2.2.4 Western Blot Analysis

Primary cortical neuronal cultures and rat brain samples were lysed in RIPA buffer
(25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate,
0.1% SDS) containing 1% (v/v) mammalian protease inhibitor cocktail and 1%
(v/v) phosphatase inhibitor cocktail from Sigma and Thermo Scientific, respec-
tively. Proteins in cell lysates were separated on a SDS-PAGE. After proteins were
transferred to a nitrocellulose membrane, the membrane was then blocked in block-
ing buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20, 5% milk) for 1.5 h at
room temperature. After blocking, membranes were incubated overnight with fol-
lowing antibodies: GRP78 and p-IRE1(1:2,000; abcam), CHOP and p-Perk (1:1,000;
Cell Signaling) and caspase-12 (1:500; Santa Cruz Biotechnology). Membranes
were then incubated with ECL horseradish peroxidase-conjugated anti-rabbit or
anti-mouse IgG (1:3,000; GE Healthcare, UK) for 90 min in room temperature.
GAPDH (1:3,000; Cell Signaling) and b-actin (1:2,000; Santa Cruz Biotechnology)
were used as internal controls. Extensive washes with blocking buffer were per-
formed between each step. The protein immunocomplex was visualized using ECL
detection reagents purchased from Thermo Scientific. Quantitative Western blot
results were obtained by densitometric analysis using Image Processing and
Analysis in Java (Image J).