Biology Reference
In-Depth Information
8. The particles should be fi nely dispersed on the macrocarrier.
Clumps will lead to cell death and lower transformation rates.
Presence of small amounts of water in the resuspended parti-
cles will lead to clumping during drying. It is not necessary to
put the macrocarriers in a 37 °C incubator when the relative
humidity in the lab is low.
9. If the rupture disk holder is not securely tightened, the disk
will slip out at low pressure. This may still result in transforma-
tion, but the yield of expressing cells may be lower.
10. Optimal incubation time depends on the proteins that need to
be expressed. First signals can usually be observed after a few
hours. Expression can be stable for several days, but fungal
growth often appears on the second day of incubations. A typi-
cal incubation is between 16 and 24 h, in other words from the
afternoon of the fi rst day to the morning or afternoon of the
next.
11. The epidermal cells can be identifi ed by their well-defi ned out-
lines. Further into the sample are the mesophyll cells which
were typically broken during the peeling process. It is impor-
tant that the epidermis is closest to the cover slip to ensure
best image quality.
12. It is usually best to perform the initial scan with illumination
for an organelle marker since they tend to express very well
and result in bright fl uorescent images. For example, a CFP
marker is easy to detect while the weak autofl uorescence of the
cell walls will allow for orientation during the scanning pro-
cess. Scanning for RFP is usually more diffi cult since the tissue
emits very little signal at this wavelength and makes orienta-
tion diffi cult. Autofl uorescence of the tissue, e.g., resulting
from damage during preparation, can sometimes be confused
with actual signal from the FPs. However, most autofl uores-
cence appears with several fl uorescence fi lters, whereas FP
fl uorescence can be detected only by the appropriate fi lter set.
13. It is best to follow a regular path to ensure that all cells are
examined. For example, move the stage up in a straight line to
observe the left edge of the sample, shift over to the right by
approximately one fi eld of view, and move down until the
lower edge of the tissue is reached.
14. In most cases, distribution of transformed cells is not uniform
but occurs in patches. It is often possible to identify patches
with 10-20 or more cells in a small area. These patches are
very convenient since it is easy to move to neighboring cells
even with a high-magnifi cation objective.
15. Depending on the intracellular distribution of the fl uorescent
signal, it may be necessary to focus further into the cell. For
example, the nucleus of these epidermal cells is usually found
attached to the back wall, about 70
μ
m into the cell. Due to
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