Histology fish accessory kit

Ultra-bright telomere spots were detected by FISH on paraffin-embedded tissue slides. The Histology FISH Accessory Kit (DAKO) was used following the manufacturer’s instructions. A telomere-specific PNA-Cy3-labelled probe (DAKO) was used for the detection of ubs42 . Tissue images were captured using a CCD camera with focus motor (Photometrics SenSys camera) connected to a PC running the Cytovision image analysis system (Applied Imaging Ltd., UK) and Z-stack software.

The TMPRSS2-ERG status of samples in the validation cohort was determined by using a break-apart assay with a triple-labeled color commercial probe (Kreatech Diagnostics, The Netherlands). The probe detects the deletion between TMPRSS2 and ERG at 21q22. The FISH assay was carried out on 4 μm formalin-fixed, paraffin-embedded tissue sections after deparaffinization which were then pretreated using a commercial tissue section kit for paraffin-embedded tissue (Histology FISH Accessory Kit, Dako). The probe mix was applied and denatured at 80°C for 5 minutes before hybridization at 37°C overnight using a Dako hybridizer. The slides were counterstained with DAPI (4′,6-diamidino-2-phenylindole) from the Histology FISH Accessory Kit. Results were visualized using a 100x oil immersion objective on a Nikon Eclipse 90i fluorescent microscope (Nikon Corp., Japan) equipped with appropriate filters. For each sample, 25 non-overlapping nuclei in cancer areas were evaluated for deletion of the TMPRSS2 (21q22) gene region associated with TMPRSS2-ERG. In order to compensate for nuclear truncation, the cut-off level for an informative result was defined as loss of the TMPRSS2 (21q22) gene region at least 80% of tumor cell nuclei.

FISH assay was performed on tissue microarrays using the Histology FISH accessory kit (K579911-2, Dako, Agilent Technologies, Santa Clara, CA, USA) according to manufacturer instructions. Thirty-eight tumors from the ICGC cohort were analyzed. Each case was represented by three spots 4 µm-thick and 1 mm in diameter. FISH assay was performed using a commercially available MYOCD FISH probe labeled in spectrum orange and a chromosome 17 control probe labeled in FITC (EG-MYOCD-CHR17-20ORGR, Empire Genomics, Williamsville, NY, USA). MYOCD and control probe enumeration was performed with a Nikon Eclipse 90i fluorescent microscope with appropriate filters. Pictures were captured using a Pannoramic 250 Flash II Digital Slide Scanner and analyzed with the Pannoramic Viewer (3DHISTECH Ltd., Budapest, Hungary). A case was considered interpretable when almost 80% of cells presented a signal for both probes. A loss was defined when only one copy of MYOCD was observed in the majority of cells; a normal status was when two copies of MYOCD were detected in the majority of cells; a gain or polysomy was when 3 to 5 copies of MYOCD or both MYOCD and the control probe were detected; and amplification was when the number of MYOCD signals was equal to or greater than 6, mainly when clustered signals were observed.

FISH was performed using the Histology FISH Accessory kit (DAKO, Glostrup, Denmark) on 5-μm sections from FFPE samples, as recommended by the manufacturer. The probes consisted of a chromosome-17 centromeric probe (CEP17 Spectrum Green ™ probe, Vysis, Abbott Molecular, Des Plaines, Illinois, USA) and an in-house generated bacterial artificial chromosome (BAC) probe located in the amplified region of chromosome 17 (clone RP11-1055B8). The BAC probe was labeled with SpectrumOrange™ using a nick translation kit (Abbott Molecular, Des Plaines, Illinois, USA) and the specificity of the BAC probe was verified on leucocytes by metaphase FISH.
Images were collected with the BioView Duet™ System (BioView Ltd, Rehovot, Israel). Signals from both probes were scored in about 100 non-overlapping nuclei with malignant morphology in the infiltrating tumor zone (as selected by the pathologist). Adjacent fibroblasts or lymphocytes and residual normal breast tissues were used as internal control. For each tumor, the mean number of signals corresponding to the RP11-1055B8 probe was calculated. The ratio between the RP11-1055B8 probe and the centromeric probe was also evaluated for individual nuclei and the percentage of the nuclei with a ratio ≥3 was calculated for each TNBC. FISH slides were analyzed blinded to the CGH results.

Where IHC analysis was positive, FISH was performed on 3 μm FFPE tissue sections using the ALK FISH DNA break-apart Probe, Split Signal (Dako) according to the manufacturer’s recommendations. Slides were pretreated at 98 °C in solution for 10 min and digested with pepsin for 3 min at 37 °C using the histology FISH accessory Kit (Dako). Slides were incubated for 18 h at 45 °C with ALK probes diluted at 1:10, and had been previously denatured for 5 min at 85 °C. Slides were then washed and dehydrated before counterstaining and application of mounting medium. Slides were analyzed with a Zeiss AxioImager Z1 fluorescence microscope (Labexchange, Burladingen, Germany). Slides were analyzed independently by two pathologists. A minimum of 100 nuclei were scored and cases were considered positive when more than 15% of cells displayed split signals.

FISH assay was performed using the Histology FISH accessory kit (Dako) as previously described according to the manufacturers' instructions [14] (link). Interphase molecular cytogenetic studies using a commercially-available ALK probe (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe, Abbott Molecular) were performed on a 4-µm paraffin-embedded section. Nuclei were scored for non-rearranged patterns (red and green signals overlapping or close together) and unbalanced patterns (split red and green signals or single red signals) using a Nikon Eclipse 80i fluorescent microscope with appropriate filters. Pictures were captured using a Hamamatsu C4742-95 CCD camera and analyzed with the Genikon software (Alphelys). The positive threshold was defined as more than 15% of signals split and/or isolated red signal as previously described [15] (link) in 100 tumor cells.

Fluorescent in situ hybridization (FISH) for TP53 with TexasRed-labeled 17p13.1 (TP53 gene) probe and FITC-labeled CEP17 (chromosome 17 centromere) probe (p53 kit, Cytocell-Aquarius, Cambridge, UK) was performed on 7μm-thick tissue sections of 8 primary RCCs and of 8 metastatic tumor samples corresponding to the 8 primary RCCs. Slides were processed using the Histology FISH accessory kit® (Dako, Denmark) according to the manufacturer's recommendations. The slides were hybridized overnight with the specific TP53 probe. Samples were analyzed with an AxioImager.M1 epifluorescence microscope (Zeiss, Hamburg, Germany). Images were captured with a x63 oil immersion objective and were analyzed using the Isis® software (METAsystems, Altlussheim, Germany). At least 200 intact, non-overlapping nuclei were scored for each sample. The numbers of TP53 alleles (red fluorescence) and CEP17 (green fluorescence) were recorded in each cell, to determine the prevalence of each genetic abnormality. The percentage of TP53 copy-numbers (monosomy, disomy or trisomy) was the number of nuclei with 1, 2 or 3 TP53 alleles among the 200 tumor nuclei analyzed. Results were expressed as mean ± standard error of the mean (SEM).