To correlate UNR association with the chromosomal content (DNA FISH) or with the poly(A) tail of mRNA (RNA FISH), sequential fluorescent labeling was performed using 6-μm tissue sections, prepared from formalin-fixed paraffin-embedded human Hep-3B xenografts [10 (link)]. Firstly, UNR immunodetection was performed, images were captured and coordinates of each image were recorded to ensure repositioning of the slide to the same area after FISH assay. Secondly, interphase DNA or RNA FISH detection was performed using the same slide; the images were captured using the repositioning function of the microscope (Zeiss Axioplan 2 fluorescence microscope, Zeiss, Jena, Germany).
DNA FISH targeted the short arm of chromosome 6 (chromosome region 6p25) (SpectrumRed probe, Abbott France, Rungis, France) and the long arm of chromosome 11 (chromosome region 11q13.3) (SpectrumGreen probe, Abbott France) and was performed as previously described [13 (link)]. RNA FISH targeting polyadenylated mRNA was also performed as described [6 (link)]. At each step, slides were mounted with Vectashield antifade medium containing 4’,6-diamidino 2-phenylindole (DAPI) (Vector Laboratories, Laboratoires Eurobio/Abcys, Les Ulis, France).
DNA FISH targeted the short arm of chromosome 6 (chromosome region 6p25) (SpectrumRed probe, Abbott France, Rungis, France) and the long arm of chromosome 11 (chromosome region 11q13.3) (SpectrumGreen probe, Abbott France) and was performed as previously described [13 (link)]. RNA FISH targeting polyadenylated mRNA was also performed as described [6 (link)]. At each step, slides were mounted with Vectashield antifade medium containing 4’,6-diamidino 2-phenylindole (DAPI) (Vector Laboratories, Laboratoires Eurobio/Abcys, Les Ulis, France).
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