Rabbit anti wt1

Frozen kidney sections were permeabilized, blocked, and incubated with goat anti-Synaptopodin (Santa Cruz), Nephrin (R&D Systems), rabbit anti-WT1 (Abcam), Ki67 (Abcam), p-Stat3, p-Src, p-Erk, p-H2AX, or NF-κB p65 (Cell signaling), then incubated with Alexa 488- or 594-labeled donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories) or fluorescein labeled Lotus Tetragonolobus Lectin (LTL) (VECTOR) and mounted in DAPI-Fluoromount-G Clear Mounting Media (SouthernBiotech). For antigen retrieval when staining for WT1, p-Stat3, p-Src, and p-H2AX, sections were heated in pH = 6 citrate buffer and permeabilized with Triton x-100 0.2% in phosphate buffered solution (PBS) for 5 minutes, followed by incubation with blocking solution of 5% BSA in PBS at room temperature for 30 minutes. Sections were imaged using an Axio Imager 2 and Observer microscopes (Zeiss). The ratio of cells expressing the protein of interest to total (DAPI + ) cells was calculated for each image. Fluorescence intensity for glomerular staining was measured using Image J and at least 160 glomeruli were evaluated in each group.

Staining for WT1 and CD68 were performed on acetone-fixed frozen sections (7μm) after endogenous biotin was blocked using a biotin blocker system (DAKO, Carpinteria, CA). To detect Type 1 Collagen (Col-1), formalin-fixed sections (5μm) were deparaffinised and antigen retrieval performed by boiling sections for 10 minutes in 10 mM sodium citrate buffer (pH 6.0). Sections were then incubated with 10% normal horse serum followed by 60 minute incubation with primary antibodies: rat anti-mouse CD68 (ABD Serotec Inc., Oxford, UK), rabbit anti-WT1 (Abcam, Cambridge, UK), rabbit anti-Col-1 (Abcam, Cambridge, UK) or concentration-matched isotype negative control. Endogenous peroxidase activity in the sections was quenched with H₂O₂ prior to application of biotinylated anti-rat IgG or anti-rabbit IgG (BD Biosciences, Pharmingen). A Vector stain ABC kit (Vector Laboratories Inc) was applied to the tissue followed by DAB solution (DAKO).