Figure 2. Co-localization and interaction between Pax6 and SPARC. Fluorescence microscopy images of the retinal region of murine eyes sections showing co-localization and interaction of Pax6 and SPARC. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. The sections were immunostained with anti-Pax6+anti–SPARC. A: The green signal was detected by fluorescein isothiocynate (FITC)-conjugated secondary IgG that shows the expression of Pax6. B: the red signals detected by tetra-methyl rhodamine isothiocynate (TRITC)-conjugated secondary IgG show the expression of SPARC. C: Merged images show the co-localization of Pax6+SPARC. Arrows in the insets (D, E, F) show the magnified images of the expression and co-localization. Negative control for immunostaining. G: without anti-Pax6 primary antibody but with FITC-conjugated secondary antibody no signals were observed, H: without anti-SPARC primary antibody but with TRITC-conjugated secondary antibody no signals were observed, I: blue signal by staining with DAPI shows the entire population of the cells. J: Co-immunoprecipitation assay between Pax6 and SPARC that is immunoprecipitated (IP) with anti-Pax6 and immunoblotted (IB) with anti-SPARC. The next lane of the blot shows negative control of IP experiment with protein-G slurry without antibodies. K: Immunoblotting (IB) with anti-Pax6 for immunoprecipitated (IP) with anti-SPARC that confirms their physical interaction. The next lane of the blot shows negative control of IP experiment with protein-G slurry with anti-Pax6 antibody, immunoblotting with anti-Pax6 using liver as non-Pax6 expressing tissue, and eye lysate as a positive control showing specificity of anti-Pax6.