Species specific secondary antibody

The histological section of renal (3 μm) was used for immunohistochemical staining of VDR (catalog no. sc-13133, Santa Cruz, USA) and Beclin1 (catalog no. BN500-249, Novus, USA) proteins. After antigen retrieval with sodium citrate buffer, sections were incubated with a primary antibody, and then the specimen was incubated with a species-specific secondary antibody (Millipore, USA). Subsequently, the sections were visualized with diaminobenzidine (Maixin, China) and the nuclei were counterstained with hematoxylin. The staining intensity was observed under a light microscope and assessed by IHC Profiler in Image J software (Media Controbernetics Inc, Rockville, USA), and 20 consecutive non-repetitive fields of view were taken from the kidney of each rat.

Histological section of renal (3μm) were used for immunohistochemical staining of podocin (Abeam, UK), nephrin (Abeam, UK), desmin (Abcam, UK), VDR (Santa Cruz, USA), WT1 (Abcam, UK), P62 (Cell signaling, USA) and Beclin-1 (Novus, USA) proteins. After antigen retrieval, sections were incubated with primary antibody, and then the specimen was incubated with a species-specific secondary antibody (Millipore, USA). Subsequently, the sections were visualized with diaminobenzidine (Maixin, China) and the nuclei were counterstained with hematoxylin. The staining intensity was observed under light microscope, and 20 consecutive non-repetitive fields of view were taken from the kidney of each rat. Analyzed with Image-Pro plus 6.0. The final assessment for each sample was the average of the analysis from 20 glomerulus. Pathological evaluation was performed blindly.