Anti her2

Cells were cultured on chamber slides and processed for immunofluorescent staining as described earlier^{33 (link)}. Briefly, upon 3 washes with PBS, cells were permeabilized with 0.5% Triton X-100 for 15 min at room temperature, and the blocking step was performed using a blocking solution containing 3% NFDM, 1% BSA, 0.1% Triton X-100 in PBS for 30 min at room temperature. Chamber slides were next incubated overnight at 4 °C with a mixture of the rabbit mAb anti-HER2 (Abcam, Cambridge, UK)(0.5 µg/ml in the blocking solution) and either mouse mAb anti-HER2 clone 70.27.58 (0.5 µg/ml in blocking solution) or clone 70.21.73.67 (0.5 µg/ml in blocking solution). After 3 washes with PBS, the chamber slides were incubated for 1 h at room temperature with secondary anti-mouse IgG-AF488 (Thermo Fisher Scientific, Waltham, MA, USA) and anti-rabbit IgG-AF594 (Abcam, Cambridge, UK). Unbound antibodies were removed by 3 washes with PBS, followed by mounting the chamber slides with the mounting medium containing DAPI for nuclear staining. Control slides with a single antibody, as well as secondary antibody controls, were performed in parallel to exclude possible unspecific reactions. The slides were observed under the Nikon Eclipse Ti-2U Fluorescent microscope (Tokyo, Japan).

After the breast cancer tissues and cells were harvested, the RIPA lysis buffer (Beyotime Biotechnology) with PMSF was conducted to isolate the total protein. Then a BCA Protein Assay Kit (Beyotime Biotechnology) was carried out to quantify the protein concentrations. A total of 25 μg proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electro-transferred into PVDF membranes (Millipore, Massachusetts, USA). The above membranes were blocked using 5% skimmed milk and then incubated with the primary antibodies, such as anti-TFAP2C (also named AP2 gamma, 1/25000 dilution, Abcam), anti-HER2 (1/1000 dilution, Abcam), anti-Akt (1/500 dilution, Cell Signaling Technology), anti-p-Akt (Ser473, 1/500 dilution, Cell Signaling Technology), anti-ERK (1/1000 dilution, Abcam), anti-p-ERK (ERK1/2, Thr202/Tyr204, 1/1000 dilution, Santa Cruz Biotechnology) and anti-GAPDH (1/1000 dilution, Abcam) overnight at 4 °C. Then the membranes were incubated with a secondary antibody (1/2000 dilution, Abcam) at RT for 1 h. All the bands were visualized using enhanced chemiluminescence (ECL) reagent (ThermoFisher Scientific).

To maintain the three-dimensional structure of the organoids, BME-organoid mixture was aspirated from the 24-well plates gently and completely, then it was embedded in Collagen (Biocoat, Corning, NY, USA). Collagen was added to pre-warmed 48-well plates (Nest, Wuxi, Jiangsu, China) and solidified at 37 °C for 2 h. All samples were fixed in 4% paraformaldehyde before embedded in paraffin. The protein expression was assessed following a two-step method. Rabbit anti-ER antibody (1:5), anti-PR (1:100), anti-HER2 (1:1000), and anti-Ki67 (1:200) were purchased from Abcam (Cambridge, MA, UK). Anti-CK5/6 antibody, Anti-p63, Anti-Calponin and the DAB kit were purchased from Zhongshan Goldenbridge Biotechnology Company (Beijing, China).