Rabbit anti rat pax2 polyclonal antibody

For western blot analysis, tissue samples were homogenized and sonicated prior to lysis. Cells and tissue samples were lysed for 1 h in a RIPA lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40,) in the presence of 1 μl/ml phenylmethylsulfonyl fluoride (PMSF). The lysed samples were centrifuged at 15,000 rpm for 20 min and boiled for 10 min. Protein concentrations were measured with a BCA Protein Assay Kit (Beyotime). Protein samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech). The PVDF membranes were blocked with TBST buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.2% Tween 20) containing 5% non-fat dry milk for 0.5 h. After washing with TBST, the membranes were probed with each antibody as indicated. The following antibodies were used; rabbit anti-rat Pax2 polyclonal antibody (1∶400, Santa Cruz), rabbit anti-rat CD24 polyclonal antibody (1∶200, Santa Cruz), anti-mouse IgG and anti-rabbit IgG secondary antibody (1∶1000, Cell signaling technology). Immunoreactive proteins were visualized using an enhanced chemiluminescence (ECL) system (Forevergen). Triple replicates were performed for each experiment. The following antibodies were used: mouse anti-rat β-actin polyclonal antibody.

For the indirect immunofluorescence analysis, cells grown on coverslips were fixed with 4% para-formaldehyde for 20 min, followed by permeabilizing with 0.1% Triton X-100 for 30 min (For Pax2 detection). After blocking in 3% bovine serum albumin (BSA), cells were probed with each antibody as indicated. The following antibodies were used: rabbit anti-rat Pax2 polyclonal antibody (1∶400, Santa Cruz), rabbit anti-rat CD24 polyclonal antibody (1∶200, Santa Cruz) and FITC-labeled anti-rabbit secondary antibody (1∶1000, Cell Signaling Technology) [25] (link). The immunofluorescence images were recorded with a fluorescence microscope (Nikon 80i Microscope).