Vectastain elite abc system

Protein samples were run on Bio-Rad TGX Stain-Free precast gels and visualized on the UV setting in the BioRad Chemidoc MP Imaging System to estimate total protein per lane. Blocking and antibody incubations were done in NAP blocker^TM (G-Biosciences). The mouse anti-VDR (Abcam) was used 1:10,000 and incubations were done overnight at 4°C. For detection, the blots were incubated overnight at 4 °C with anti-mouse biotin (Jackson Laboratories) followed by anti-mouse streptavidin-HRP (Vectastain Elite ABC system, Vector Laboratories). Molecular weight markers are Precision Plus Protein Kaleidoscope Standards (BioRad). Protein detection was achieved with femtoCHROMO^TM–HRP (G-Biosciences) and visualized using the colorimetric setting in the BioRad Chemidoc MP Imaging System.

After deparaffinisation, the sections (4 μm) were cut and incubated with primary antibodies against Ki‐67 (ab16667; Abcam, Cambridge, UK), heparan sulfate 6‐O‐sulfotransferase 1 (HS6ST1, bs‐10701R; Bioss Antibodies, Woburn, MA, USA), endothelial cell‐specific molecule 1 (ESM 1; ab103590; Abcam, Cambridge, UK), and the endothelial cell marker CD31 (M0823; Dako, Santa Clara, CA, USA). The target proteins were visualised using the VECTASTAIN Elite ABC system (Vector Laboratories) or the secondary antibodies (Alexa Fluor 488 and 568, Invitrogen) and Hoechst nuclear stain. The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.

Zinc and FFPE samples were deparaffinized followed by the antigen retrieval procedure. Human FFPE samples in TMA as well as mice samples for staining of HO-1 and P-Erk1/2 were retrieved by high pressure cooking (HPC; 2100-Retriever, EMS, Hatfield, PA, CatNo. 62706) in citrate buffer (pH 6.0) for 1 hour; Zinc fixed xenograft samples were retrieved in DMSO for 3 minutes for CD86 and Gr-1 staining. Acetone was used after fixation of sections to detect NK antigen. After washing with 1x PBS, slides were blocked with 7% horse serum (Normal Horse Serum, VectorLabs, Peterborough, UK). Primary antibodies were applied overnight at 4¼C. The following day, slides were washed once in 1x PBS, blocked with H₂O₂, and then washed three times in 1xPBS for five minutes per wash. Biotin-labeled secondary antibodies were applied for an hour at room temperature. VECTASTAIN Elite ABC System was used to enhance the signals (VectorLabs). DAB substrate (VectorLabs) was used to develop the reactions, followed by slide dehydration, mounting, and analysis by light microscopy. Microphotographs were taken under 100-200x magnifications in the light microscope (Nikon). Immunostainings were quantified by evaluating percent of positive staining or number of cells or intensity of staining per field of view using Soft View software. Details are provided in Figure legends.

Urinary albumin and creatinine were determined by using mouse albumin ELISA and creatinine companion kits (Philadelphia, PA USA) following manufacturer’s protocols. Serum levels of creatinine were determined by using a kit from Biovision (Milpitas, CA). For immunohistochemical staining, formalin-fixed and paraffin-embedded renal tissues were cut into 4- to 5-μm sections. The slides were stained with anti-collagen IV (Cell Signaling) or F4/80 antibody (Serotec) using VECTASTAIN Elite ABC system (Vector Lab) as described previously [25 (link)]. For immunofluorescence staining: frozen kidney samples were sectioned and fixed in cold acetone. The slides were stained with anti-WT1 antibody (Novus) and then with fluorescence conjugated secondary antibodies (Life Technologies). After washing, slides were incubated with DAPI for 5 minutes and coverslipped. In addition, kidney electron microscopy (EM) was performed by using the service from the Imaging Center at University of Kentucky.