ALP activities were expressed as units per liter (U/L). Immunohistochemistry staining of colon and distal ileum Immunohistochemistry was performed on Dasatinib groups A, B, E, and F of the distal ileum and colon to confirm the translation of CXCR4 mRNA to protein. Specimens were routinely fixed in 10% formalin in the immediate postoperative period and paraffin-embedded within the first 6 hours after procurement. After sectioning samples (5 ��m), slides were dried overnight at 37��C and were deparaffinized with xylene. The sections were treated with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 98��C for 20 minutes, and the slides were washed 2 �� 5 minutes in TBS plus 0.025% Triton X-100 with gentle agitation. Blocking was done in 10% normal serum with 1% bovine serum albumin in TBS for 2 hours at room temperature.
Samples were incubated overnight at 4��C with a primary antibody diluted in TBS with 1% bovine serum albumin at a dilution of 1:100. The next day, sections were rinsed 2 �� 5 minutes with tris-buffered saline (TBS) 0.025% Triton with gentle agitation. The slides were incubated in 0.3% H2O2 in TBS for 15 minutes. Sections were labeled with a goat antirabbit IgG horseradish peroxides (HRP), 200 ��g/0.5 mL (Santa Cruz Biotechnology) and developed with 3,3��-diaminobenzidine (brown-colored staining) and counterstained with hematoxylin (blue color staining). Results Characterization of CXCR4 siRNAI, II/dextran-spermine nanoparticles In this study, transmission electron microscopy was applied to determine the morphology of nanoparticles 8 hours after preparation.
Nanoparticles were formed by mixing CXCR4 siRNAI, II with dextran-spermine at various weight-mixing ratios (siRNA/dextran-spermine) from 1:1 to 1:5. Nanoparticles formed at a ratio of 1:5 repelled one another and did not form aggregation. CXCR4 siRNAs/dextran-spermine nanoparticles formed small, round particles with good repelling properties against each other (data not shown). The particle size of nanoparticles was automatically calculated by particle size analysis NANOPHOX (0128 P) (Nanophox, Clausthal-Zellerfeld, Germany). The average size of nanoparticles was 54.74 �� 0.00 nm. This experiment was done three times and each time was repeated 40 times (Figure 1). The zeta potential or surface charge of nanoparticles (mV) was automatically calculated and found to be 39.
7 �� 0.2, and zeta deviation (mV) was found to be 6.52 �� 0.6 (Figure 2). Figure 1 Particles size distribution of CXCR4 siRNAI, II/dextran-spermin q3 = Fractional volume density distribution. Figure 2 Zeta potential of CXCR4 siRNAI, II/dextran-spermine. CXCR4 expression by RT-PCR one-step Total RNA was isolated from the liver of six groups of animals 35 days after CT26.WT cell injection. RT-PCR was performed on the Entinostat real-time PCR machine RotorGene 3000 (Corbett), and delta-delta Ct method was used to analyze CXCR4 and housekeeping gene expression.