Workshop proceeding - final.pdf - Faculty of Information and ...

triphenyltetrazolium chloride (TTC) solution (1%) for 30 min in phosphate buffer at 37℃. Sections
were fixed overnight in 4% paraformaldehyde for contrast enhancement between stained and
unstained tissue. TTC stained the viable tissue with red, while the necrotic tissue remained discolored.
The sections were then placed between two cover slips and digitally photographed using a Nikon
coolpix S10 camera, and quantified with the weight respectively. The area of irreversible injury (TTC
negative) is presented as a percentage of the area(the irreversible injury area/the total weight of
ventricles).
2.7.Measured the density of capillary
The streptavidin peroxidase (SP) immunohistochemical method was used to detect the expression
of factor VIII in myocardial infarction margin areas. The dilution of factor VIII rat monoclonal
antibody (Santa Cruz, CA, USA) was 1:100. The procedure was performed according to the
manufacturer’s instructions.The positive cells were identified, counted and analyzed under the
inverted phase-contrast microscope (Olympus, Japan) in 3 different fields(0.1mm 2 ) at 400
magnification using Image-proplus 6.0 software.Results were normalized by arbitrarily setting the
total tube area of control to 100%.
2.8. Real time fluorescent quantitation PCR (RT-PCR)
After ligation, myocardial tissues were harvested at the times indicated, washed twice with icecold
phosphate-buffered saline (PBS) and collected by centrifugation. Total RNA was isolated using
the Trizol reagent (MRC, USA) according to the manufacturer's instructions. Total RNA (5 µL) was
converted to complementary DNA (cDNA) using Revert Aid First Strand cDNA Synthesis Kit. A
5µL aliquot of the resulting cDNA was used as template for PCR amplification with the following
primers: mini-TrpRS, P1 (forward, 5’- CCC TGC TGC ACT CCA CCT T-3’), P2 (reverse, 5’- ACG
CAT GCT TAT TGA CCT TG-3’); mini- TyrRS, P3 (forward, 5’- CAT CTG ATG AAT CCT ATG
GTT -3’), P4 (reverse, 5’- GGA TCA CAA ACT CGG ACT TA-3’); β-actin, P5 (forward, 5’-GCC
AAC ACA GTG CTG TCT -3’), P6 (reverse, 5’-AGG AGC AAT GAT CTT GAT CTT -3’). The
amplifications were performed by an initial denaturation (94 °C for 2 min), followed by 45 cycles of
denaturation, annealing and extension (94 °C for 20 s, 54 °C for 20 s, 72 °C for 30 s), and a final
extension (72 °C for 5 min). The transcript of β-actin was also amplified by RT-PCR from the same
cDNA template and was used as an internal control. All the primers were designed and synthesized by
Genepharma (Shanghai, China). The identity of each PCR product was confirmed by DNA sequencing.
The pictures were scanned and analyzed by the Gel Doc 1000 gel imaging system.
2.9.Statistical analysis
Results are expressed as mean ± standard deviation. Comparison of means was performed by
means of the analysis of variance procedure (Student–Newman–keuls test, SPSS 13.0 for Windows).
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