Renal tissues specimens were obtained from 4% paraformaldehyde fixative, and then dehydrated and embedded in paraffin. The embedded tissues were sliced into 4-μm-thick sections, which were stained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 5 min at room temperature, then stained with eosin (Sigma-Aldrich) for 3 min at room temperature, and observed under a DMi8 optical microscope (Leica, Hamburg, Germany) at ×200 magnification.
Sirt6 expression in the renal tissues from the rats with or without DN were examined by IHC staining. The sections were dewaxed using two types of xylene solution, hydrated by ethanol at various gradients and antigen retrieval was achieved by quenching with endogenous peroxidase. The sections were then washed with PBS for 3 times and incubated with primary Sirt6 antibodies (1:100; ab135566, Abcam, Cambridge, MA, USA) at 4°C overnight and then washed with PBS. The sections were then incubated with secondary antibody biotin-conjugated goat anti-rabbit IgG (Proteintech Group, Inc./Thermo Fisher Scientific; SA00004-2; 1:200) at 25°C for 30 min. Subsequently, diaminobenzidine (DAB) was used as a chromogen and hematoxylin was used to re-dye the sections, at 25°C for 3 min. The stained sections were examined using a DMi8 optical microscope (Leica) in randomly selected sections at ×200 magnification.
Sirt6 expression in the renal tissues from the rats with or without DN were examined by IHC staining. The sections were dewaxed using two types of xylene solution, hydrated by ethanol at various gradients and antigen retrieval was achieved by quenching with endogenous peroxidase. The sections were then washed with PBS for 3 times and incubated with primary Sirt6 antibodies (1:100; ab135566, Abcam, Cambridge, MA, USA) at 4°C overnight and then washed with PBS. The sections were then incubated with secondary antibody biotin-conjugated goat anti-rabbit IgG (Proteintech Group, Inc./Thermo Fisher Scientific; SA00004-2; 1:200) at 25°C for 30 min. Subsequently, diaminobenzidine (DAB) was used as a chromogen and hematoxylin was used to re-dye the sections, at 25°C for 3 min. The stained sections were examined using a DMi8 optical microscope (Leica) in randomly selected sections at ×200 magnification.