Fig. 1
From: Use of multicolor fluorescence in situ hybridization to detect deletions in clinical tissue sections
Design of flanking probes to detect tumor suppressor gene deletions illustrated using an example of the PTEN gene. a Genomic deletion sizes of chromosome 10 derived from public domain datasets. Each horizontal thin red line represents an individual deletion and the best location for the selection of probes flanking PTEN (red vertical line) is just outside the minimal region of PTEN deletion (green and blue vertical lines). b Position of flanking probes WAPAL (green box) and FAS (aqua box) selected because they map outside the PTEN (red box) minimal region of deletion shown in b. The centromere probe (magenta box) is included to help determine the number of chromosome 10 s in the sample. c Schematic diagram to illustrate how a four-color probe design can be used to recognize truncation losses due to sectioning in a nucleus (gray spheres). In this illustration, the PTEN probe (red spots) is flanked either side of the gene by two probes (blue and green spots) on the two black wavy lines representing chromosome 10, which is enumerated by the CEP10 magenta probe. When there is loss of red PTEN with both blue and green flanking probes it indicates that sectioning (shown schematically as a dashed blue line in middle nucleus) has removed nuclear material that contained part of chromosome 10. However, when an actual interstitial deletion of PTEN is present, both flanking probes are retained (shown in right nucleus), indicating that the loss of a PTEN signal is due to an actual deletion. d Schematic three-dimensional representation to illustrate how four-color FISH is used to identify various truncated nuclei in a FFPE section. The nuclei are depicted as uniform pale blue spheres that are distributed randomly throughout the cuboid three-dimensional volume that represents part of a 5 µm section (left). In each nucleus the same spot colors as a are used to depict PTEN (red), the flanking probes (blue and green), and CEP10 (magenta). The thick blue dashed double arrowed vertical line points to the interface of two adjacent sections illustrating how truncated nuclei may be identified. It can be seen that there are six nuclei (red arrows) which will be recognizable with this probe set as truncated nuclei by the concurrent losses of a red PTEN probe with both green and blue flanking probes
