Anti mouse pax2

Immunofluorescence was performed on cells grown on coverslips and in matrigel. The cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min and permeabilized with 0.5% Triton X-100 for 30 min for cells grown on coverslips or 0.2% Triton X-100 for 10 min for cells grown in matrigel. After blocking in 5% goat serum, cells were probed with each antibody as follows: anti-mouse PAX2 (1:500, Santa Cruz), anti-mouse PAX8 (1:200, Santa Cruz), anti-rabbit E-cadherin (1:200, Abcam) and anti-rat CD44 (1:200, Abcam). Alexa Fluor goat anti-mouse IgG (1:1000), goat anti-rabbit IgG (1:500) and goat anti-rat (1:500; all from Molecular Probes, Carlsbad, CA) were used as secondary antibodies. The cells on coverslips were then mounted to microscope slides using Vectashield hard set mounting medium with DAPI (Vector Laboratories, Burlingame, CA) and the immunofluorescence images were visualized and analyzed using an inverted fluorescence microscope (Axioskop 2 MOT plus, Zeiss) and Axiovision software.

Cellular proteins were recovered from cell cultures using the M-PER mammalian protein extraction reagent (Thermo Fisher, Waltham, MA). Cell proteins were separated and transferred to nitrocellulose as previously described [53 (link)], and then probed with the following antibodies: anti-mouse PAX2 (1:20000, Santa Cruz Biotechnology, Dallas, Texas) and Anti-rabbit CD44 (1:500), Anti-rabbit E-cadherin (1:10,000), Anti-rabbit vimentin (1:10,000), Anti-rabbit Snail (1:10,000), Anti-rabbit cytokeratin 19, anti- rabbit OVGP (1:1000) (all from Abcam, Cambridge, UK). Anti-rabbit (1:10,000, Abcam) or anti-mouse (1:10,000, Sigma-Aldrich) horseradish peroxidase-conjugated antibodies were used as secondary antibodies. Immunoreactive proteins were detected using chemiluminescence (Clarity Western ECL Substrate; Bio Rad) and the blots were imaged using FluorChem FC2 (Alpha Innotech). The densitometric analyses of protein abundance were performed using ImageJ software (NIH Image).