Fig 1.
(A) Schematic of FLASR (ZZ-rsEGFP2tandem) bound to an immunoglobulin protein. (B-D) Maximum intensity projections of confocal z-stacks of methanol fixed mammalian CV-1 cells immunolabelled with antibodies against β-actin (B), vimentin (C) and α-tubulin (D). Subsequently, purified FLASR (red) was used to decorate the primary antibodies. Nuclei were stained with DAPI (blue). Scale bars: 50 μm.
Table 1.
Binding parameters of the interaction between FLASR and polyclonal secondary antibody solutions of different mammalian species.
FLASR was immobilized on a Ni2+-chelator sensor chip. Concentrations of 7.8 nM to 4.0 μM of the indicated IgG were used to monitor the association and dissociation. Data were analyzed with a simple 1:1 Langmuir interaction model to determine rate constants for the association and dissociation, which were then used to calculate the indicated dissociation constants.
Fig 2.
RESOLFT nanoscopy of methanol fixed cells.
Comparison of RESOLFT super-resolution microscopy and the corresponding confocal microscopy images of CV-1 cells decorated with primary antibodies against vimentin (A), α-tubulin (B) and the nuclear pore complex protein Nup153 (C). (D) Line-profiles of the fluorescence intensities recorded between the arrowheads in (A-C), as indicated (confocal: light blue; RESOLFT: red). The line profiles in (1–3) are averaged across five adjacent line profiles that were perpendicular across the respective filament. The distance between two adjacent line profiles was the edge length of one pixel. Scale bars: 1 μm.
Fig 3.
RESOLFT super-resolution image of an entire CV-1 cell.
The cell was decorated with primary antibodies against α-tubulin and FLASR. Scale bar: 5 μm.
Fig 4.
Immunolabelling with an M-rsEGFP2tandem fusion protein.
Maximum intensity projections of confocal microscopy z-stacks of methanol fixed CV-1 cells immunolabelled with antibodies against β-actin (A) and vimentin (B). The purified recombinant fusion protein M-rsEGFP2tandem was used to decorate the primary antibodies (red). Nuclei were labelled with DAPI (blue). Scale bars: 50 μm.