Tumor-bearing kidneys were harvested and immediately fixed with 10% formalin in PBS for 24 hours. Tissue was then washed and stored in 70% ethanol before being embedded in paraffin and sectioned to 4 μm thickness. Sections were baked for 30 min at 60°C to melt excess paraffin. Sections were stained with hematoxylin and eosin. Immunohistochemistry staining for anti–cyclin D1 (Neomarkers, no. RM-9104), anti–Ki-67 (Biocare, no. CRM325), and phospho-Ser807/811–pRb (Cell Signaling, no. 9308) was performed on a Bond III (Leica Biosystems) with the Bond Polymer Refine Detection Kit (Leica Biosystems, #DS9800). Antigen retrieval was performed using Bond Epitope Retrieval Solution 2 for 20 min (cyclin D1 and Ki-67) or Epitope Retrieval Solution 1 for 30 min (phospho-Ser807/811–pRb). Sections were incubated for 30 min with primary antibody diluted in Bond Primary Antibody Diluent followed by incubation for 10 min with HRP-conjugated secondary antibody. Sections were then incubated with chromogen 3,3′-diaminobenzidine for 5 min to visualize staining. Sections were counterstained with hematoxylin and dehydrated in graded ethanol and xylene. Slides were digitized using a ScanScope XT (Leica Biosystems), and representative images were obtained using the Indica Labs Halo platform.

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