Identifying Subcellular Protein Localization with Fluorescent Protein Fusions After Transient Expression in Onion Epidermal Cells - Plant Cell Morphogenesis: Methods and Protocols - page 80

3. Put open agar plate with onion peels on second shelf from the
bottom.
4. Close particle gun and pull vacuum to about 27 mmHg. Press
“Fire” switch until rupture disk breaks. Vent the chamber and
remove the agar plate.
5. Repeat the procedure ( steps 2 - 4 ) with the same agar plate for
the second macrocarrier disk of this sample.
6. After the second shot with the same sample, wrap agar plate
with parafi lm and store in a dark place at room temperature
until the next day ( see Note 10 ).
7. Repeat as needed for all samples.

3.5 Mounting
Epidermal Peels
for Microscopy

1. Place clean cover slip on a paper towel and add small drops of
water all over its surface.
2. With fi ne forceps, pick up the epidermal peel and place it on
the cover slip in same orientation as on the agar (in other
words, the outer surface that was down on the agar plate
should also be down on the cover slip). Do this with a slight
rolling movement (similar to rolling out a rug) to prevent the
formation of air bubbles between the cover slip and the tissue.
Put as many epidermal strips on the cover slip as possible.
3. Put a few drops of water on the back of the epidermal peels.
Gently lay a microscope slide on the cover slip and pick it up
immediately; the cover slip will adhere to the slide.

1. Orient yourself with a low-magnifi cation objective (10× or
20×) under bright-fi eld illumination and focus on the epider-
mal cell layer ( see Note 11 ). Move sample so that the lower
left corner of the tissue piece is in view.
2. Switch over to fl uorescent illumination ( see Note 12 ) and scan
over the tissue piece to identify transformed cells ( see Note 13 ).
On an inverted microscope, the position of transformed cells
can be marked on the upper side of the slide with a small dot
from a felt-tip marker to make it easier to return to them later.
On upright microscopes, the coordinates can be noted down
from the stage markings ( see Note 14 ).

3.6 Microscopy:
Identifying
Transformed Cells

3.7 Microscopy:
High- Magnifi cation
Imaging

1. Switch to a high-magnifi cation objective suitable for observa-
tion of subcellular structures (e.g., 63×/1.4 oil immersion).
Focus on the marker signal. The highest quality images can be
obtained in the cytoplasm right behind the outer plasma mem-
brane ( see Note 15 ). This area also has the advantage that
many organelles can be observed at the same time.
2. Switch the fl uorescent fi lters to observe the signal from the
unknown protein. Ideally, the signal of the marker and the
unknown should be of roughly equal intensity to avoid