The paraffin-embedded kidney sections were first dewaxed and subjected to antigen retrieval. Then, they were blocked with 5% bovine serum albumin for 30 min at room temperature. The sections were next incubated with a mixture of rabbit anti-Sirt6 polyclonal antibody (1:100, Thermo Fisher Scientific, USA) and mouse anti-WT1 monoclonal antibody (1:100, Novus, USA) overnight at 4°C.The sections were stained with a mixture of Alexa Fluor 488, Donkey anti-mouse IgG (HL) (1:200, Jackson Immuno Research Laboratories, USA) and Alexa Fluor 594, Donkey anti-Rabbit IgG (HL) (1:200, Jackson Immuno Research Laboratories, USA) as the secondary antibodies at 37°C for 60 min in the dark. The nuclei were counterstained with DAPI (Antgene, China) for 5 min.
The cell-climbing films (cells growing on glass slides) were fixed with 4% paraformaldehyde for 30 min at 4°C and stained with rabbit anti-Sirt6 polyclonal antibody (1:100, Thermo Fisher Scientific, USA) overnight at 4°C. The films were then incubated with Alexa Fluor 594, Donkey anti Rabbit IgG (HL) (1:200, Jackson Immuno Research Laboratories, USA) at 37°C for 60 min. The nuclei were counterstained with DAPI (Antgene, China) for 5 min. A confocal microscope (Olympus, Japan) was used to record all of the microscopic images.
The cell-climbing films (cells growing on glass slides) were fixed with 4% paraformaldehyde for 30 min at 4°C and stained with rabbit anti-Sirt6 polyclonal antibody (1:100, Thermo Fisher Scientific, USA) overnight at 4°C. The films were then incubated with Alexa Fluor 594, Donkey anti Rabbit IgG (HL) (1:200, Jackson Immuno Research Laboratories, USA) at 37°C for 60 min. The nuclei were counterstained with DAPI (Antgene, China) for 5 min. A confocal microscope (Olympus, Japan) was used to record all of the microscopic images.