Four m thick sections of formalin
Four-μm thick sections of formalin-fixed paraffin-embedded tissue samples from uterine cervical cancers were cut with a microtome and dried overnight at 37 °C on a silanized-slide (Dako, Carpentaria, CA, USA). The protocol of the universal Dako Labeled Streptavidin–Biotin kit (Dako) was followed for each sample. Samples were deparaffinized in xylene at room temperature for 30 min, rehydrated with graded ethanol and washed in phosphate buffer saline (PBS). The samples were then placed in 10 mM citrate buffer (pH 6.0) and boiled in a microwave for 10 min for epitope retrieval. Endogenous peroxidase activity was quenched by incubating tissue sections in 3% H2O2 for 10 min. The primary antibodies, rabbit EphB4 (sc-5536, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit ephrinB2 (sc-1010, Santa Cruz) were used overnight at 4 °C at dilutions of 1:100 and 1:75, respectively. The slides were washed and biotinylated anti-rabbit secondary antibody (Dako) was applied for 30 min. After rinsing in PBS, streptavidin-conjugated horseradish peroxidase (Dako) was added on the slides and kept for 30 min. Slides were then treated with the chromogen 3, 3′-diaminobenzidine (Dako) for 5 min and rinsed in PBS. After counterstaining with Mayer\'s hematoxylin samples were dehydrated in graded ethanol, cleared in xylene and cover-slipped with a mounting medium, Entellan New (Merck, Darmstadt, Germany). Rabbit pre-immune animal serum (Dako) was used for negative controls instead of the primary antibody for EphB4 or ephrinB2. All sections of immunohistochemical staining for EphB4 and ephrinB2 were evaluated in a semi-quantitative fashion according to the method described by McCarty et al. , which considers both the intensity and the percentage of (Z)-4-Hydroxytamoxifen stained at each intensity. Intensities were classified as 0 (no staining), 1 (weak staining), 2 (distinct staining), 3 (strong staining) and 4 (very strong staining). For each stained section, a value-designated histoscore was obtained by application of the following algorithm: histoscore=Σ(i+1)×Pi, where i and Pi represent intensity and percentage of cells that stain at each intensity, respectively, and the corresponding histoscores were calculated separately. Total RNA was extracted with the acid guanidinium thiocyanate–phenol–chloroform method . Total RNA (3 μg) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (MMLV-RT, 200 U/μl, Invitrogen, Carlsbad, CA, USA) and the following reagents: 250 mM Tris–HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2, 0.1 M dithiothreitol, 10 mM deoxynucleotide (deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycystidine) tri-phosphates (dNTPs) mixture and random hexamers (Invitrogen) at 37 °C for 1 h. The reaction mixture was heated for 5 min at 94 °C to inactivate MMLV-RTase. Real-time PCR reaction was performed with a Takara Ex Taq R-PCR kit, version 1.0 (Takara, Otsu, Japan), using a smart cycler system (Cepheid, Sunnyvale, CA, USA). The reaction solution (25 μl) contained Takara Ex Taq HS (5 U/μl), 10×R-PCR Buffer, 250 mM Mg++ solution, 10 mM dNTP mixture, SYBR Green I (1:1000 dilution; Cambrex Bio Science Rockland, Inc. Rockland, ME, USA) and 20 μM of primers for the EphB4 gene, 2501–2625 in the cDNA (EphB4-S: 5′- ACGGACAGTTCACAGTCATC-3′ and EphB4-AS: 5′- GCAACATCCTAGTCAACAGC-3′) and for the ephrinB2 gene, 679–785 in the cDNA (ephrinB2-S: 5′- CAACATCCTCGGTTCCGAAG-3′ and ephrinB2-AS: 5′- CCTCTTGCTGAAGTACCGGA-3′) with the transcribed total RNA from the tissue and a serially diluted standard template. The real-time PCR reactions were initially denatured by heating at 95 °C for 30 s, followed by 40 cycles consisting of denaturation at 94 °C for 10 s, annealing at 55 °C for 5 s and extension at 72 °C for 20 s. A strong linear relationship between the threshold cycle and the log concentration of the starting DNA copy number was always shown (correlation coefficient>0.99). Quantitative analysis was performed to determine the copy numbers of each sample.