Figure 5
Co-localization of VHA-subunits with the tonoplast marker γ-Tip and calreticulin, a marker for the ER. Confocal images represent single images of isolated maize root cells which were immuno-probed with antibodies directed against marker polypeptides of the vacuolar membrane (γ-Tip) and endoplasmatic reticulum (calreticulin) and simultaneously treated with anti-VHA-antibodies. Secondary fluorescently labelled antibodies used were anti-rabbit-FITC (A), anti rabbit-Cy3 (B, E, H), anti-mouse-FITC (D, G, J) or anti-guineapig-Cy5 (K). Images were colour coded with Adobe Photoshop. Scalebars are 10 μm. In each row, the immuno-decoration with the marker, with the VHA-subunit specific antibody and the superposition of both is shown. (A-C) Root cell labelled with γ-Tip (A) and VHA-A (B). Note the complete co-localisation of the both markers on the tonoplast of small vacuoles (C). (D-F) Double-staining with calreticulin (D) and VHA-A. (G-I) Double-labelling of a root cell with calreticulin (G) and VHA-E (H) reveals a similar result as with VHA-A. Tonoplast labelling and ER-staining are distinct. (J-L) Co-labelling with calreticulin (J) and VHA-aN-term reveals a complete co-localisation of the two signals on the ER.