Image pro 6

Formalin-fixed Paraffin-embedded (FFPE) cancer tissue was delivered from clinical institutions from all over Germany. FFPE tissue was cut into small pieces (2 µm) on a slide and dehydrated using first a xylene washing step subsequent flowed by a series of ethanol steps (100%, 96%, 70%). After drying the slide at room temperature slides were incubated with sodium thiocyanate followed by a wash step using distilled water. Subsequently, slides were incubated with pepsin and hydrochloric acid, washed using distilled water and dried at room temperature. Probes (PathVysion HER-2 DNA Probe Kit II, Abbott Inc.) were hybridized at 37 °C in a wet chamber overnight. Washing of slides was performed in 2x saline-sodium citrate (SSC) buffer and DAPI counterstaining was conducted. Images were taken using fluorescence microscope (Axioskop 2, Zeiss Inc.) using a graded filter (Filter Set 23 (488023-0000-000), emission: 515–530 nm + 580–630 nm, Zeiss Inc.), recording HER2 gene signals, CEN17 signals and a small subset of DAPI signals at once. Images were captured at a magnification of 40x and processed using the Image-Pro 6.0 software (Media Cybernetics) and saved in JPEG file format with a size of 1200 × 1600 pixel.

Tumors and normal tissues used for immunohistochemistry (IHC) staining, which was manipulated as described previously^{24 (link)}, were obtained from the patients (without radiotherapy or neoadjuvant chemotherapy treatment) with breast cancer at the First Affiliated Hospital of Chongqing Medical University. The primary antibody against p-ATM (1:200, Abcam), anti-PFKP (1:200, CST), anti-CS (1:200, CST), and secondary antibody (1:1000, ZSBIO) were used. The images were captured and evaluated by the Image-Pro 6.0 software (Media Cybernetics, Rockville, MD, USA) and scored by mean optical density (density/area). p-ATM, PFKP, and CS staining were scored into five intensities: 0, no staining; 1 + , 1–25%; 2 + , 26–50%; 3 + 51–75%; and 76–100%.