Fluoromount

The cells were blocked for 1 h (BSA in PBS, pH 7.4) and then incubated with primary antibodies overnight at 4 °C or for 2 h at room temperature, respectively. The immune complexes were identified with the respective specific secondary antibodies for 1 h at room temperature. Paraffin-embedded human renal biopsy sections were deparaffinized, rehydrated, treated for antigen retrieval and incubated in blocking solution prior to the incubation with primary antibodies at room temperature (RT) for 1 hour or at 4 °C overnight according to different antibodies.
The following primary antibodies were used: mouse anti-human TLR2 mAb (HyCult Technologies, Plymouth Meeting, PA), rabbit anti-human inhibin A pAb (Novus Biologicals,), mouse anti-human CD133 mAb (Miltenyi Biotec), rabbit IgG cleaved caspase-3 (Asp175) mAb (R&D Systems). The following secondary antibodies were used: Alexa Fluor 555 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, and Alexa Fluor 488 goat anti-mouse IgG1 (all from Molecular Probes). All cells and sections were counterstained with To-pro-3 (Molecular Probes, Eugene, OR, USA) and mounted in Fluoromount (Leica, Wetzlar, Germany). Negative controls were prepared with irrelevant antibody. The stained cells were viewed under the Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscope using ×40 and ×63 objective lenses.

Paraffin-embedded human RCC sections were deparaffinized, rehydrated, treated for antigen retrieval, and incubated in blocking solution prior to incubation with primary antibodies at room temperature (RT) at 4 °C overnight. The immune complexes were identified with the respective specific secondary antibodies for 2 h at room temperature. The following primary antibodies were used: rabbit polyclonal CYP1B1 (ab137562, Abcam), mouse anti-human monoclonal CD133/2 (pure, 120-001-247, Miltenyi Biotec). The following secondary antibodies were used: Alexa Fluor 555 goat anti-rabbit IgG, Alexa Fluor 488 goat anti-mouse IgG. Human CD133 (Prominin-1) mouse anti-human monoclonal Ab (TMP4) PE (#12-1338-42,Invitrogen) was used to perform direct immunolabeling. All cells and sections were counterstained with To-pro-3 (Molecular Probes, Eugene, OR, USA) and mounted in Fluoromount (Leica, Wetzlar, Germany). Nuclei were counterstained with DAPI (Thermo Fisher, Waltham, MA, USA, dilution 1:1000). Images were acquired under the Leica TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscope using ×20 objective lenses, and images were acquired with Leica Application Suite XV3.7.6.25997 software (Leica microsystems CMS GmbH).