Cells and two different tumour sites using a DNeasy Tissue kit

Cells and two different tumour sites using a DNeasy Tissue kit (Qiagen, UK). PCR was conducted in a Primus 96 Plus PCR Thermocycler (MWG AS Biotech, Ebersberg, Germany) with the following primers (Invitrogen): UBC promoter: forward 59-GAACAGGCGAGGAAAAGTAGTCC-39; reverse 59ACCAGGGCGTATCTCTTCATAGC-39; product size: 1091 bp. Reactions were set up using 3 mM 25033180 MgCl2 (Invitrogen, UK), 0.2 mM each dNTPs (Invitrogen, UK), 16PCR buffer (Invitrogen, UK), 0.5 mM of each forward and backward primer (Invitrogen, UK), 100 ng DNA, 0.25 ml Taq (5 U/ml) (Invitrogen, UK) and the final volume adjusted to 50 ml with dH2O. Template DNA was initially denatured at 95uC for 5 minutes, followed by 30 cycles of denaturation at 95uC for 45 seconds, annealing at 60uC for 45 seconds and primer extension at 72uC for 1 minute. A final 10-minute incubation at 72uC allowed for complete extension. PCR products were analysed on 0.8 agarose gels.ImmunohistochemistryTumour tissue was fixed in paraformaldehyde and paraffin waxembedded, before being cut into sections 4 mm in thickness. Sections were taken through histoclear (National Diagnostics, Georgia, USA) and a series of decreasing concentrations of ethanol to dehydrate them. Sections were stained with haematoxylin and eosin to observe tissue morphology. For immunohistochemical analysis of luciferase expression, sections were incubated in 3 hydrogen peroxide to block endogenous peroxidases and rinsed in ethanol solutions of decreasing concentration to rehydrate the sample. Sections were incubated in 0.01 M sodium citrate buffer and treated with avidin and biotin (Vector Laboratories, CA, USA). Sections were blocked in horse serum and then incubated overnight at 4uC with a 1:50 dilution of rabbit monoclonal antiluciferase antibody (Santa Cruz Biotechnology, Santa Cruz, USA). The next day sections were incubated with a 1:1000 dilution of biotin-conjugated horse anti-rabbit immunoglobulin (Vector Labs) followed by addition of the Vectastain ABC complex (Vector Labs) according to the manufacturer’s instructions. 79831-76-8 Colour was developed by incubation with DAB substrate (Vector Labs) for 5 minutes. Slides were stained with haematoxylin and dehydrated by rinsing in a series of ethanol solutions of increasing concentration, before being mounted and visualised using an LEICA DM4000 B microscope with a LEICA DFC420 camera inverted microscope. Image acquisition and analysis was performed using Leica LAS software, Lite version.Southern Blot AnalysisFor DNA analysis, total cellular or tumour DNA (collected from two different tumour sites) was extracted using a DNeasy Tissue kit (Qiagen, UK). The isolated DNA was quantified using a NanoDrop ND1?000 spectrophotometer (Labtech International Ltd, Ringmer, UK). For Southern analysis, total tumour DNA (15 mg) was digested with the single cutting restriction enzyme (SpeI) and separated on 0.8 agarose gels (20 V, 20 mA overnight) and blotted onto nylon membranes (Hybond XL, Amersham plc, Little Chalfont, UK). A 408 bp DNA fragment derived from the restriction digest of a segment of the 1313429 kanamycin Eliglustat supplier region, which is common to all plasmids, using enzyme AlwNI, was labelled with 32P (Rad-Prime labelling kit, Invitrogen, UK) and used as a probe. The hybridization was performed in Church buffer (0.25 M sodium phosphate buffer (pH 7.2), 1 mM EDTA, 1 BSA, 7 SDS) at 65uC for 16 h. For the replication-dependent restriction assay, 15 mg of total tumour DNA was digested with SpeI and further digested wi.Cells and two different tumour sites using a DNeasy Tissue kit (Qiagen, UK). PCR was conducted in a Primus 96 Plus PCR Thermocycler (MWG AS Biotech, Ebersberg, Germany) with the following primers (Invitrogen): UBC promoter: forward 59-GAACAGGCGAGGAAAAGTAGTCC-39; reverse 59ACCAGGGCGTATCTCTTCATAGC-39; product size: 1091 bp. Reactions were set up using 3 mM 25033180 MgCl2 (Invitrogen, UK), 0.2 mM each dNTPs (Invitrogen, UK), 16PCR buffer (Invitrogen, UK), 0.5 mM of each forward and backward primer (Invitrogen, UK), 100 ng DNA, 0.25 ml Taq (5 U/ml) (Invitrogen, UK) and the final volume adjusted to 50 ml with dH2O. Template DNA was initially denatured at 95uC for 5 minutes, followed by 30 cycles of denaturation at 95uC for 45 seconds, annealing at 60uC for 45 seconds and primer extension at 72uC for 1 minute. A final 10-minute incubation at 72uC allowed for complete extension. PCR products were analysed on 0.8 agarose gels.ImmunohistochemistryTumour tissue was fixed in paraformaldehyde and paraffin waxembedded, before being cut into sections 4 mm in thickness. Sections were taken through histoclear (National Diagnostics, Georgia, USA) and a series of decreasing concentrations of ethanol to dehydrate them. Sections were stained with haematoxylin and eosin to observe tissue morphology. For immunohistochemical analysis of luciferase expression, sections were incubated in 3 hydrogen peroxide to block endogenous peroxidases and rinsed in ethanol solutions of decreasing concentration to rehydrate the sample. Sections were incubated in 0.01 M sodium citrate buffer and treated with avidin and biotin (Vector Laboratories, CA, USA). Sections were blocked in horse serum and then incubated overnight at 4uC with a 1:50 dilution of rabbit monoclonal antiluciferase antibody (Santa Cruz Biotechnology, Santa Cruz, USA). The next day sections were incubated with a 1:1000 dilution of biotin-conjugated horse anti-rabbit immunoglobulin (Vector Labs) followed by addition of the Vectastain ABC complex (Vector Labs) according to the manufacturer’s instructions. Colour was developed by incubation with DAB substrate (Vector Labs) for 5 minutes. Slides were stained with haematoxylin and dehydrated by rinsing in a series of ethanol solutions of increasing concentration, before being mounted and visualised using an LEICA DM4000 B microscope with a LEICA DFC420 camera inverted microscope. Image acquisition and analysis was performed using Leica LAS software, Lite version.Southern Blot AnalysisFor DNA analysis, total cellular or tumour DNA (collected from two different tumour sites) was extracted using a DNeasy Tissue kit (Qiagen, UK). The isolated DNA was quantified using a NanoDrop ND1?000 spectrophotometer (Labtech International Ltd, Ringmer, UK). For Southern analysis, total tumour DNA (15 mg) was digested with the single cutting restriction enzyme (SpeI) and separated on 0.8 agarose gels (20 V, 20 mA overnight) and blotted onto nylon membranes (Hybond XL, Amersham plc, Little Chalfont, UK). A 408 bp DNA fragment derived from the restriction digest of a segment of the 1313429 kanamycin region, which is common to all plasmids, using enzyme AlwNI, was labelled with 32P (Rad-Prime labelling kit, Invitrogen, UK) and used as a probe. The hybridization was performed in Church buffer (0.25 M sodium phosphate buffer (pH 7.2), 1 mM EDTA, 1 BSA, 7 SDS) at 65uC for 16 h. For the replication-dependent restriction assay, 15 mg of total tumour DNA was digested with SpeI and further digested wi.