Fig. 4. KNOLLE protein immunolocalisation in developing root hairs of 35S::KN seedlings. Seedlings were grown on 1% agar with 0.5x MS salts and 1% sucrose. (A) Dark-field, (B) epifluorescence microscopy, (C-Q) optical sections, confocal laser-scanning microscopy; n, nucleus; pm, plasma membrane. (A-M,O-Q) 35S::KN, (N) wild-type control. (B,C,E-G,N) Anti-KN antiserum (Cy3, red) and DAPI (blue) staining. (D,H-M) Double labelling with anti-KN antiserum (Alexa-m488, green) and anti-PM-ATPase monoclonal antibody (Cy3, red) and DAPI (blue) staining. (O-Q) Membrane labelling of live seedling roots with lipophilic dye FM4-64. The surface of root hairs and the root is delineated by broken lines in (E-G,K-O,Q). (A-D) Overviews. (A) Seedling root with root hairs of increasing age. (B-D) KN accumulation in root hairs: (B) young root hair tip (boxed) of similar age as those in (E,H-J); (C) old root hairs; boxed area is enlarged in G and similar region is shown in Q; (D) old root hairs; boxed area is shown in (K-M). Surface of root hairs is delineated by white lines in (C,D). (E-G) KN accumulation in young (E), mid-age (F) and old (G) transgenic root hairs. (H-M) Co-localisation of KN and PM-ATPase in young (H-J) and old (K-M) root hairs. (H,K) KN staining, (I,L) PM-ATPase staining, (J,M) overlays; pink colour in J is due to additional DAPI nuclear signal. Note co-localisation of the two antigens in old root hair (K-M), which is partly collapsed owing to fixation-induced plasmolysis. (N) Young root hairs of wild-type control showing no KN signal; compare with B,E. (O-Q) Endomembrane staining of young (O), mid-age (P) and old (Q) root hairs. Compare (O,P) with strong KN accumulation in root hair tips (E,H) filled by vesicles, ER and Golgi (see Fig. 5K,L). In old root hairs (Q), FM4-64 staining resembles the KN staining (C,G).