Envision system hrp labelled polymer anti rabbit k4003

TMA tissue sections (2.5 μm) were deparaffinized and epitopes were retrieved using TEG buffer, as previously described [60 (link)]. TMA sections were stained with rabbit monoclonal ERG antibody (2805–1, Epitomics) in a 1:150 dilution in TEG. Secondary staining was performed using the EnVision + System (HRP Labelled Polymer Anti-Rabbit K4003, DakoCytomation). For all cores, ERG immunoreactivity (0,no staining; 1, moderate; 2, strong) was evaluated and scored by two independent observers (SH and ASL) as described above. Positive staining was used as a proxy for ERG fusion status as previously described [45 (link), 46 (link)].

Formalin-fixed paraffin-embedded (FFPE) blocks from PDX tumors or breast cancer organoids were sectioned at 4 µm onto Superfrost Plus slides. Immunohistochemistry was carried out using the DAKO Autostainer Link 48. Sections underwent dewaxing, heat-induced antigen retrieval using DAKO Target Retrieval Solution (S1699) at 98 °C for 30 min, endogenous peroxidases were quenched by applying Dako Real Peroxidase Blocking solution (S2023) for 10 min, followed by Dako Serum Free Protein Block (X0909) for 30 min. Then, primary antibody incubation using FGFR4 (sc-136988, Santa Cruz Biotechnology, 1:300 dilution) or Ki-67 antibody (9027, Cell Signaling Technology, 1:300 dilution) was followed by the Dako Envision + System – HRP Labelled Polymer Anti-Rabbit (K4003) secondary antibody incubation system. Lastly, sections were counterstained with Dako Automation Hematoxylin Histological Staining Reagent (S3301). For the Ki67 staining analysis, at least 10 field of vision images per sample were taken with ImageScope viewer and positive areas quantified using the ImageJ software. For FGFR4, the sample was considered positive if more than 1% of cells exhibited staining.