Combined Ihc And Cish Of Her2 In Breast Cancer

The protocol described here allows immunohistochemis-try (IHC) and chromogenic in situ hybridization (CISH) to be combined, such that genetic alternation and protein expression were evaluated using an ordinary light microscope. This technique can have many uses. It can be used, as in this experiment, to evaluate the genetic modification of the gene and the resulting alternation of gene expression;1-4-1 or IHC can be used simply as marker to differentiate different cell type.

The FFEP sections were prepared and pretreated with pretreatment solution as previously described. For immu-nohistochemistry, the sections were preblocked with CAS-Block for 10 min, then incubated with rabbit antibody to HER2 (Zymed) for 30 min. After washing

Fig. 3 HER2 IHC/CISH in 4-mm-thick paraffin section of breast carcinoma open biopsy. Tumor cells with clusters of highly amplified HER2 gene and strong IHC staining can be seen. The arrows point to normal HER2 signals in cells of a normal breast epithelium, which is negative for IHC staining. (View this art in color at www.dekker.com.)

Fig. 3 HER2 IHC/CISH in 4-mm-thick paraffin section of breast carcinoma open biopsy. Tumor cells with clusters of highly amplified HER2 gene and strong IHC staining can be seen. The arrows point to normal HER2 signals in cells of a normal breast epithelium, which is negative for IHC staining. (View this art in color at www.dekker.com.)

in PBS/T, the slides were incubated with AP-labeled goat antirabbit IgG (Zymed) at 1:100 dilution in antibody diluent for 30 min and washed with PBS/T. Then, the color of AP was developed with Vector-blue (Vector). The sections were then digested with Digest-All 3 and in situ hybridization was carried out according to the protocol described in the previous section using digox-igenin-labeled HER2 probe (Zymed). For detection, the slides were first incubated with CAS-Block for 10 min, and then incubated with FITC-antidigoxigenin (Roche Applied Science) at 1:500 dilution in antibody diluent for 60 min. After washing in PBS/T, the slides were further incubated with HRP-anti-FITC (Zymed) for 60 min and washed in PBS/T. Then, the color of HRP was developed with DAB (Zymed). The slides were counterstained with hematoxylin and mounted in Glycerogel (DAKO).

The IHC results and CISH of highly amplified HER2 gene can be viewed at 10 x objective. The tumor cells with cluster of amplified HER2 gene usually are also strongly positive for the HER2 IHC (Fig. 3). The normal cells are negative of HER2 IHC and the nuclei contain only one or two CISH signals for the HER2 gene. Using conventional bright-field microscopy, the non-amplified HER2 signal could be observed with 40 x objective. However, because of the reflective nature of DAB, the signal can be observed at 10x using phase-contrast microscopy.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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