Arabidopsis cells were fixed in 4% formaldehyde (freshly prepared from paraformaldehyde (Sigma) in stabilizing buffer: 50 mm PIPES, 5 mm MgSO4, 5 mm EGTA pH 6.9) for 1 h, then washed three times with stabilizing-buffer for 10 min on ice and once with PBS (6.5 mm Na2HPO4 2H2O, 1.5 mm KH2PO4, 2.5 mm KCl, 0.14 m NaCl pH 7.1). The samples were dehydrated in a graded ethanol/PBS series on ice and embedded in polyethylene glycol 400 distearate : 1-hexadecanol (10 : 1 mixture, Aldrich) at 37°C according to Vitha et al. (1997) . After hardening the wax at room temperature, sections 4–5 μm thick were cut using a rotary microtome, and the ribbons were placed on slides coated with either glycerol-albumin or poly l-lysine, expanded by adding a drop of distilled water, and dried overnight at room temperature. To remove the wax, the dried samples were treated with decreasing ethanol/PBS series and washed with PBS. The samples were incubated with a mouse monoclonal anti-HA antibody (diluted 1 : 400 in stabilizing buffer containing 2% BSA, fraction V, fatty acid-free, Boehringer) for 1 h at room temperature in a moisture chamber, then washed three times with PBS. Subsequently the samples were similarly treated with FITC-conjugated goat anti-mouse IgG (Sigma, F-0257, dilution 1 : 200) for 1 h, washed three times with PBS and mounted with SlowFadeTM (Molecular Probes) to visualize the HA antigen in HiA-GUS-transformed cells. For double labelling, samples of SKP1-HiA-transformed cells were treated in a similar manner with a rabbit polyclonal anti-AtSKP1 antibody (dilution 1 : 250) and mouse anti-HA IgG, then incubated with FITC-conjugated goat anti-mouse IgG (1 : 200) in combination with a CyTM 3-conjugated anti-rabbit IgG (H + l, Jackson ImmunoResearch Laboratories, 1 : 400) as described above. The rabbit polyclonal anti-AtSKP1 antibody was raised against a C-terminal AtSKP1 peptide (KNDFTPEEEEEVRRE) that was marked by an N-terminal cysteine facilitating its conjugation to carrier proteins. This AtSKP1 peptide shared at least nine amino-acids with other known members of the SKP1-LIKE protein family in Arabidopsis. The polyclonal anti-AtSKP1 antibody was affinity purified using the AtSKP1 peptide linked to Affi-Prep 10 support (Bio-Rad) as described ( Harlow & Lane 1988). In control experiments, the antibodies were either independently or sequentially applied to the cell sections in order to examine and exclude a possible cross-reaction between monoclonal and polyclonal IgGs. The specificity of immunodetection was further tested by omitting the treatments with primary anti-HA and anti-AtSKP1 antibodies before the application of FITC-and CyTM 3-conjugated second antibodies. In competition experiments the primary polyclonal anti-AtSKP1 and monoclonal anti-HA antibodies were blocked with an excessive amount of SKP1 and HA peptides, respectively, overnight at 4°C before applying them onto the sections to confirm that these treatments abolish the signal in immunodetection. Fluorescence images were examined with a Leica DMRB epifluorescence microscope and photographed with a Hitachi HV-C20 camera controlled by a diskus microscopic computer programme (Carl H. Hilgers, Technisches Büro, Köln, Germany).