After washing in 0. 4�� SSC 0. 3% NP 40 selleck chemicals Seliciclib pH 7 for 2 min at 73 C and in 2�� SSC 0. 1% NP 40 pH 7 7. 5 for 1 min at room tem perature, cell nuclei were counterstained with DAPI. Examina tion was done at a fluorescence microscope with slider module. Image stacks at 0. 9 um intervals were taken of at least three representative fields per cell line. Image stacks were converted into 3D view by AxioVision software. For each cell line, the gene and chromosome specific signals were counted per indivi dual cell nucleus. The mean and standard deviation of the gene and chromosome specific signals of counted cell nuclei were calculated for each cell line. The FISH ratio was calculated for each analyzed cell nucleus and thereof the mean and standard deviation was calcu lated for each cell line.
True gene specific amplification was considered at a FISH ratio of 2. The FISH proce dure and quantification has previously been published by us for evaluation of Aurora A and other gene copy numbers in tissue specimens. Indirect immunofluorescence and evaluation of mitoses Cells were grown on coverslips, fixed in 2% PFA, washed in PBS and permeabilized in 0. 5% Tritron X 100 in PBS. After PBS washing, cells were incubated with blocking buffer normal goat serum and 0. 3% Tritron X 100 Diluted pri mary antibodies were incubated over night at 4 C, cells were rinsed with PBS and 1,200 diluted fluorescently labelled secondary anti bodies, were incubated for 1 h at RT. After washing with PBS and distilled water, cell nuclei were counterstained with DAPI.
Note that the p53 antibody used was raised against the N terminal domain, recognizing also mutated and expressed p53 proteins. Normal bipolar mitoses were defined as mitotic cells with 2 Aurora A positive centrosomes spindle poles. Multipolar mitoses were defined as mitotic cells with 2 Aurora A positive centrosomes spindle poles. In three independent experiments, cells were screened using a x40 objective and a minimum of 100 cells were counted for the mitotic index and up to 100 mitoses per cell line were evaluated for the occurrence of multipolar mitoses. Immunoblotting Preparation of total protein and determination of pro tein concentration was performed using the Qpro teome Mammalian Protein Prep Kit and the DC Protein Assay according to the manufacturers protocols. 10 ug of total protein extracts per lane were loaded onto 10% polyacrylamide gels.
Proteins were transferred onto Protran Nitrocellulose Transfer Mem brane by Semi Dry Blot. After blocking the membrane in 5% nonfat dried milk powder in Tris buffered saline with Tween Tween, pH 7. 2 GSK-3 7. 4 the primary antibodies diluted in 5% nonfat dried milk powder in TBST or 3% BSA in TBST or 5% BSA in TBST were incubated. After HRP conjugated secondary antibody incubation, the membrane was incubated with ECL reagents and exposed to autoradiography films.