RNA extraction and RT�CPCR for FGFRs and subtypes RNA from pancreatic cells or selleck tumours was extracted using TRIzol reagent (Invitrogen) according to the manufacturers’ protocol. cDNA was obtained from 5��g of total RNA, using the SuperScript III Reverse Transcriptase kit (Invitrogen) with oligos-dT primers. Semi-quantitative PCR was performed as follows: 2��l of 10 �� Buffer (Roche, Indianapolis, IN, USA), 0.2��l of Taq polymerase (5U��l?1 Roche), 0.4��l of 10mM dNTP mix (Roche), 0.1��l of each primer (100��M), 1��l of cDNA, filled to a final volume of 20��l with sterile H2O. Thermal cycling reaction using an Icycler device (Bio-Rad) was: 94��C for 2min; followed by 25�C35 cycles of 95��C for 30s, 60��C for 30s, 72��C for 45s for detection of FGFR2.
The amplified products were further extended by additional incubation at 72��C for 10min. PCR products were then loaded on a 1% agarose gel containing ethidium bromide. All quantitations were normalised to GAPDH. FGFR2 and GAPDH primers were as follows: FGFR1(IIIb) forward 5��-ACCAGTCTGCGTGGCTCACT-3��, reverse 5��-TGCCGGCCTCTCTTCCA-3�� FGFR1(IIIc) forward, 5��-GGACTCTCCCATCACTCTGCAT-3��, reverse 5��-CCCCTGTGCAATAGATGATGATC-3�� FGFR2 forward, 5��-TGACATTAACCGTGTTCCTGAG-3��, reverse 5��-TGGCGAGTCCAAAGTCTGCTAT-3�� FGFR2(IIIb) forward, 5��-GATAAATAGTTCCAATGCAGAAGTGCT-3��, reverse 5��-TGCCCTATATAATTGGAGACCTTACA-3�� FGFR2 (IIIc) forward, 5��-GGATATCCTTTCACTCTGCATGGT-3��, reverse, 5��-TGGAGTAAATGGCTATCTCCAGGTA-3�� GAPDH forward, 5��-GAAGGCTGGGGCTCATTTG-3��, reverse 5��-AGGGGCCATCCACAG-TCTTC-3��.
Immunohistochemistry Tumour tissue was fixed overnight in 10% neutral-buffered formalin at room temperature, transferred to 70% ethanol and processed for paraffin embedding using a Thermo Electron Excelsior tissue processor (Pittsburgh, PA, USA). Paraffin blocks were sectioned to 4��m thickness and placed on positively charged glass slides. Tissues were stained using a Discovery automated slide machine (Ventana Medical Systems, Tucson, AZ, USA). The primary antibodies used were Ki67 (1:750 dilution, Novocastra Laboratories, Newcastle upon Tyne, UK), and CD34 (EK-MP.12, 1:100 dilution, Accurate Chemical & Scientific Corp, Westbury, NY, USA). Secondary antibody was a goat anti-rabbit F(ab��)2 biotinylated antibody, 1:100 dilution (Jackson ImmunoResearch, West Grove, PA, USA).
Sections were counter-stained with hematoxylin to enhance visualisation of tissue morphology. General tissue morphology was evaluated using H&E staining. For TUNEL assay, tissue samples were embedded in paraffin and cut into 4-��m-thick consecutive sections. After deparaffinised in three changes of xylene and rehydrated in descending concentrations of ethanol, the sections were treated with 20��gml?1 proteinase K at 37��C for 15min and then incubated with TDT buffer containing 12.5��m biotinylated dUTP (Boehrinnger Mannheim, Mannheim, Germany) and 0.15units per ��l TDT (Takara, Kyoto, Japan) at Brefeldin_A 37��C for 70min.