Identifying Subcellular Protein Localization with Fluorescent Protein Fusions After Transient Expression in Onion Epidermal Cells - Plant Cell Morphogenesis: Methods and Protocols - page 81

bleed-through ( see Note 16 ). As a general rule, the exposure
setting should be adjusted such that the signal brightness
becomes maximal without saturating any pixels ( see Note 17 ).
Since most organelles show rapid movements, it may be nec-
essary to limit the exposure time to prevent streaking of the
organelles ( see Note 18 ). The two images for the unknown
protein and the organelle marker have to be taken in close
succession to minimize movement of the organelles between
images ( see Note 19 ).
3. To remove background noise, a second set of images should
be taken with the same exposure settings, but the excitation
light shutter closed ( see Note 20 ).

After background subtraction ( see Note 20 ) the images can be ana-
lyzed. To detect colocalization, the two images can be false-colored
and superimposed to reveal overlapping signals. The best colors to
use are red and green since their overlap will result in a different
color, yellow, that can easily be detected ( see Note 21 ). This can be
achieved by creating a new RGB image in ImageJ, converting this
into an RGB stack (with “Image > Type”) and pasting the two
images into the fi rst and second frame of the stack. Converting this
image back to an RGB image will complete the procedure.

3.8 Image Analysis

4 Notes

1. The tungsten particles can be hazardous because of their small
size. Wear gloves and possibly also a respirator.
2. Keep the centrifugal force to a minimum to prevent clumping
of the particles since this makes it diffi cult to resuspend them
later.
3. Commercial onions usually work well. Avoid previously fro-
zen onions and do not keep cut onions more than a few days.
4. The smaller size of the strips makes it easier to spread the
curved epidermal peels on the fl at agar surface.
5. Anywhere from 100 ng to 1 g of DNA per plasmid is usually

suffi cient for clearly detectable expression of fusion proteins.
The precise amount depends on the size of the plasmid, the
promoter, and nature of the fusion protein. Organelle markers
tend to express very well and require little DNA (50-100 ng).

μ

6. There is no need to resuspend the particles by vortexing.
Several tubes can be prepared in parallel.
7. There should be enough particles for three disks, but two
shots per sample normally yield suffi cient numbers of cells to
determine protein localization. The second shot can be omit-
ted too, but is handy if something goes wrong with the fi rst.