Fig. 7. Immunolocalization of p63RhoGEF in human heart and brain tissue sections. (A) Detection of p63RhoGEF protein in human brain sections. (a) Normal human cerebellum section using non-specific rabbit IgG at x40 magnification. (b) Normal human cerebellum section at x40 magnification stained with Ab699 antiserum. (c) Enlarged and focused view of the normal human cerebellum section at x100 magnification stained with Ab699 antiserum. The arrow shows one Purkinje cell surrounded by numerous round shaped cerebellar granular cells. (B) Detection of p63RhoGEF protein in human heart sections. (a) Transversal section of normal human heart at x40 magnification stained with Ab699 antiserum. The asterisk indicates the location of a blood vessel. (b) Longitudinal section of normal human heart at x40 magnification stained with Ab699 antiserum. (c) Normal human heart section using rabbit IgG at x40 magnification. (d) Normal human heart section at x40 magnification stained with Ab699 antiserum pre-incubated with the peptide 699 used to generate the anti-p63RhoGEF rabbit polyclonal antibody. (e) Longitudinal cardiac fibers from (b) shown at x100 magnification stained with Ab699 antiserum. (f) Longitudinal cardiac fibers shown at x100 magnification stained with ß-MHC-specific monoclonal antibody. (C) Double immunostaining using rabbit p63RhoGEF polyclonal antibody and either mouse anti-ß-MHC (a,b,c) or mouse anti-vinculin (d,e,f) in human left ventricle. The tissue sections were then analyzed by confocal microscopy. (a,d) p63RhoGEF exhibits a striated pattern of immunolabelling. Arrows in panel a indicate labelling organized in doublets. (b) ß-MHC immunolabelling is found in transverse A-bands (marked as `a') on both sides of the M-line (marked as `m'), which is devoid of labelling. (c) Double immunostaining analysis reveals that the immunolabelled p63RhoGEF doublets are intercalated between A-bands (potentially in the I-band; marked as `i'). (e) Vinculin immunolabelling is found in Z-disks (marked as `z') and at the level of the intercalated disk (^*). (f) Double immunostaining analysis reveals that vinculin signal is localized between immunolabelled p63RhoGEF doublet (see arrows). Bars, 10 µm.