Leica bond max automated system

PDX tissues were sectioned (5 μM) and every 20th tissue section was used for analysis. Immunohistochemistry analysis was performed using the Leica BOND-MAX automated system (Leica Microsystems, Victoria, Australia) or manually using the Dako EnVision™ detection system (Dako, California, USA). Primary and secondary antibody details and staining conditions are outlined in Supplementary Table 2. Ki67 immunohistochemical staining was performed on three representative sections from each fixed xenograft. Slides were scanned using the Aperio ScanScope AT Turbo (Leica Microsystems GmbH, Germany) and positive cells were determined using Aperio ImageScope analysis software (version 12.2).

Renal sections were analyzed for the abundance of vascular endothelial growth factor A (VEGFA; SC-152; Santa Cruz Biotechnology, Santa Cruz, CA, USA) using a biotinylated ABC kit (Vector Laboratories, Peterborough, UK) and cluster of differentiation factor 34 (CD34; ab81289; Abcam, Cambridge, UK) using a Leica Bond-Max automated system (Leica Microsystems, Wetzlar, Germany). For VEGFA, the primary antibody was diluted 1:200 in PBS and 0.05% Tween and incubated overnight at 4°C. For CD34, the primary antibody was diluted 1:1000 in Bond Primary Antibody Diluent (Leica) and incubated for 30 min at room temperature. Heat-mediated antigen retrieval was carried out for 10 min using EDTA (pH 9). Negative controls were included during each IHC run by omission of primary antibody and using a rabbit IgG control (Vector Laboratories). For both proteins, adult ovine kidney was used as a positive control.

To account for heterogeneity of prostate cancer tissue, patient specimens and first-generation PDXs were sectioned into 5 μm serial sections, with tissue pathology analyzed in every 20th section across the tumor tissue. For serially transplantable PDXs and organoids, we selected representative sections of each sample. All tissues were stained using hematoxylin and eosin (H&E) for pathological assessment. Immunohistochemistry was performed using the Leica BOND-MAX™ automated system (Leica Microsystems, Mount Waverley, Australia; Supplementary Data 9). Immunohistochemistry is performed on every fifth generation of PDX tissue.