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Blots were then washed again 3 X 5 min with TBST, followed by visualization with enhanced chemiluminescence.
Membranes were then probed with anti-rabbit or anti-mouse antibody (Amersham; GE Healthcare, Baie d'Urfé, QC, Canada) or anti-goat (Santa Cruz Biotechnology) secondary antibodies, followed by visualization with enhanced chemiluminescence (ECL; Amersham).
The proteins were transferred to a PVDF membrane by electrophoresis and probed with mouse anti-FLAG (1 1000; M2 clone, Sigma) and a goat anti-mouse-HRP secondary antibody (1 5000; Sigma), followed by visualization with enhanced chemiluminescence (Millipore).
Incubation with primary antibodies was followed by species-appropriate incubation with secondary antibodies tagged with horseradish peroxidase (1∶5000, Santa Cruz Biotechnology, Santa Cruz, CA), visualization with enhanced chemiluminescence and analysis with a Versadoc XL imaging apparatus (BioRad, Hercules, CA).
All reactions were monitored by thin layer chromatography, followed by visualization with UV and anisaldehyde-H2SO4.
The localization of the fusion proteins was determined by visualization with a confocal microscope.
Avidin-biotin peroxidase complexes (Vectastain ABC Elite, Vector Laboratories) were added followed by visualization with diaminobenzidine (Vector Laboratories).
Viability of the selected larvae was verified by visualization with a dissection microscope.
Detection of recombinant antibodies was performed by subsequent incubation of the blot with peroxidase-labeled polyclonal goat anti-mouse antibodies (Dako), followed by visualization using enhanced chemiluminiscence (ECL; GE Healthcare).
Cells were homogenized in 1% SDS, and protein extracts were analyzed by SDS-PAGE and subsequent immunoblotting with the indicated primary antibodies and horseradish-peroxidase-conjugated second antibodies followed by visualization using enhanced chemiluminescence as described previously (Webber et al., 2008).
Antibody-bound proteins were detected after treatment of membrane with horseradish peroxidase-conjugated goat anti-rabbit IgG, or rabbit anti-mouse IgG followed by visualization using enhanced chemiluminescence (ECL Pierce).
