Rabbit anti wt1 antibody

Staining for CD68 and F4/80 was performed on acetone-fixed frozen sections (7 µm) and endogenous biotin was blocked using a biotin blocker system (DAKO, Carpinteria, CA). For TLR4 and WT1 detection, formalin-fixed sections were deparaffinized and boiled 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: a rat anti-mouse CD68 antibody (ABD Serotec Inc., Oxford, UK), a rat anti-mouse F4/80 (ABD Serotec Inc.), rabbit anti-mouse-TLR4 antibody (Invitrogen, Catalogue No. 48-2300) for glomerular [22] (link), goat anti-mouse-TLR4 antibody (Santa Cruz Biotechnology, Catalogue No sc-12511) for tubular [8] (link), rabbit anti-WT1 antibody (Abcam, Cambridge, UK), or concentration-matched IgG as an isotype negative control. The sections were exposed to H₂O₂ and then incubated with biotinylated anti-rat IgG or anti-rabbit IgG (BD Biosciences, Pharmingen), or anti-goat IgG (Vector Laboratories Inc). A Vector stain ABC kit (Vector Laboratories Inc) was applied to the tissue followed by DAB solution (DAKO). Immunostaining for α-SMA was performed on formalin-fixed paraffin sections using Dako ARK Peroxidase for Mouse Primary Antibodies (DAKO) according to the manufacturer's instructions.

Immunofluorescent staining was performed as described previously [21] (link). The expression of VDR in cultured renal cells was detected using rat anti-VDR antibody (Abcam, Cambridge, UK) followed by Alexa Fluor 488 goat anti-rat IgG (Invitrogen, Carlsbad, CA). Similarly, PPARδ and COUP-TFII were detected using rabbit anti-PPARδ (Affinity Bioreagents, Golden, CO) and anti-COUP-TFII antibody (Abcam) followed by Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen). Renal expression of NOR1 was detected using mouse anti-NOR1 antibody (Abcam) followed by Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). To determine whether NOR1 was localized in mesangial cells, podocytes, proximal tubular epithelial cells, or collecting duct cells, the sections were counter-stained with rabbit anti-fibronectin antibody (Sigma-Aldrich, St. Louis, MO), rabbit anti-WT-1 antibody (Abcam, Cambridge, UK), rabbit anti-aquaporin 1 (AQP1) antibody (Millipore, Temecula, CA), or rabbit anti-AQP2 antibody (Abcam) respectively, followed by Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen). Fluorescence images were obtained using a fluorescence microscope (BX51; Olympus, Tokyo, Japan).

Mouse frozen kidney sections (7 μm) were prepared from Crb2^fl/fl mice or Crb2^fl/flpod-Cre^Tg/+ mice at 6 months of age (each n = 5). Fixation was performed in acetone at − 20 °C for 10 min. The primary and secondary antibodies were rabbit anti-NPHS2 antibody^{40 (link)} or rabbit anti-PODXL antibody^{41 (link)} or rabbit anti-NPHS1 antibody^{42 (link)} or rabbit anti-WT1 antibody (Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H&L), respectively. The images of 10 randomly selected glomeruli in each mouse were captured with an FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). The semiquantification of the expression of Nphs2, Podxl, and Nphs1 was performed as described previously^{41 (link)}. Total kidney lysates were prepared from Crb2^fl/fl mice or Crb2^fl/flpod-Cre^Tg/+ mice at 6 months of age (each n = 5) for Western blotting. The membranes were cut around 60 kDa before incubation with primary antibodies. The membrane after incubation with rabbit anti-NPHS2 antibody was stripped and then reprobed with mouse anti-β-actin antibody. Wt1-positive cells were counted in 30 glomeruli per mouse.