M3561

Total protein was extracted using lysis buffer (0.5% NP-40, 0.5% NA-DOC, 0.1% SDS, 150nM NaCl, 50 mM Tris pH 7.5, 1 mM EDTA, 1 mM NaF) supplemented with protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was measured using BCA reagent (Thermo Scientific, Rockford, IL, USA). Twenty μg of each sample was separated using 10% SDS polyacrylamide gel electrophoresis (BIO-Rad, Hercules, CA, USA) and then transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked using TBST containing 5% non-fat dry milk, then incubated with mouse-monoclonal antibodies against WT1 (1:250, catalog no. M3561, DAKO, Glostrup, Denmark), p63 (1:2000, catalog no. M7247, DAKO), p53 (1:1000, catalog no. PAb 1801, Abcam, Cambridge, UK) and β-actin (1:10000, catalog no. MAB1501R, Millipore) followed by a second incubation with peroxidase conjugated anti-mouse polyclonal antibodies (1:5000, DAKO). The antibody (anti-p63) used in this study is able to detect bands corresponding to the expected molecular weights and according to expression patterns of the various isoforms (TAp63α, TAp63γ, ΔNp63α, and ΔNp63γ). Proteins were visualized using a chemiluminescent detection system (ECL-advanced, GE healthcare UK) in ChemiDoc XRS (Bio-Rad, Italy).

Paraffin-embedded kidney sections were stained using standard histology procedures as described elsewhere [32 (link)]. Kidney injury was evaluated by Masson staining.
Immunostaining was carried out in 3 μm thick tissue sections. Antigen retrieval was performed using the PTlink system (Dako, Glostrup, Denmark) with sodium citrate buffer (10 mM) adjusted to pH 6–9, depending on the immunohistochemical marker. Endogenous peroxidase was blocked. Tissue sections were incubated for 1 h at room temperature with 4% BSA and 10% of a specific serum (depending on the secondary antibody used) in PBS to eliminate non-specific protein binding sites. Primary antibodies were incubated overnight at 4 °C. Specific biotinylated secondary antibodies (Amersham Biosciences, Amersham, UK) were used, followed by streptavidin–horseradish peroxidase conjugate and 3,3-diaminobenzidine as a chromogen. The primary antibodies used were: F4/80 [1:50 MCA497, Bio-Rad, Hercules, CA, USA] and WT-1 [1:100 M3561, Dako]. Specificity was checked by omission of primary antibodies. Quantification was made by determining in 5 to 10 randomly chosen fields (200× magnification) the total number of positive cells using Image-Pro Plus software (data expressed as the positive-stained area relative to the total area) or quantifying manually the number of positive nuclei.