2.5. CTC staining and fluorescence microscopy

Captured cells were first stained with Hoechst, antibodies against CAXII (Cedarlane Labs, 10617‐MM07‐F), CD34 (Biolegend, 343505), CD45 (Biolegend, 304008), CD66b (Biolegend, 305106), and either PD‐L1 (Abcam, ab205921) or HLA‐I (Biolegend, 311416). A donkey anti‐rabbit IgG (Biolegend 406414) secondary stain was used with the PD‐L1 antibody prior to intracellular staining. Following extracellular staining, cells were permeabilized with BD Perm/Wash buffer (BD Biosciences, 51‐2091KZ) and stained with an Alexa Fluor 790 conjugated pan‐CK (C‐11, Biolegend, 628602).

The CTCs were identified after isolation using fluorescent antibodies against both CK and CAXII, which were maintained on separate fluorescent channels to evaluate the heterogeneity of different CTC subpopulations (see Table S3 for an overview of which antibodies were used at which steps in the methods). The combination of both CAIX at the capture step, with CAXII at the identification step maximizes the specificity of CTC identification using the alternative markers, ensuring the evaluation of a population with a truly renal cancer origin.

After staining, the samples were washed three times with phosphate buffered saline (PBS), and then imaged with a 10X objective using a Nikon Eclipse Ti‐e fluorescent microscope (Nikon, Minato City, Japan) with NIS‐Elements AR 4.51.01 software (Nikon). Samples were transferred to a glass slide for high resolution imaging using a 40X apo objective. Imaging of isolated CTCs occurred within 24 h of fixation.