Biology Reference
In-Depth Information
2.3 Tools and
Microscopes
1. To dissect out old fl owers, buds, or leaves, tweezers, injection
needles, razor blades, or scalpels can be used. A good binocu-
lar, with increasing magnifi cation, is necessary: an Arabidopsis
meristem is between 100 and 300
m wide.
2. To image the meristems, we use small disposable petri dishes
or higher tissue culture dishes, which can be put under the
microscope objectives (we use a 20×, 40× or 63× water
immersion achroplan lens with a long working distance—40×
is the most versatile). We use an upright confocal laser scanning
microscope (e.g., Zeiss LSM510, 700, 710, 780 or Leica
SP5). An inverted CLSM is also usable, if the meristem is
placed deep into the medium, so that the meristem surface is
close to the medium surface, thus allowing the water column
between the lens and the meristem to be maintained over the
time of image acquisition.
μ
1. To visualize cell outlines, the FM4-64 lipophilic dye can be
used. A stock solution is prepared in water at a concentration
of 330
2.4 Dyes
g/ml and stored at −20 °C. This stock solution can
be considered to be 1× to 10× depending on the permeability
of the sample to the dye. Once diluted and in use, it can be
stored at 4 °C.
2. Propidium iodide (PI) can be used to stain cell walls. A 0.1 %
PI solution is prepared by mixing the PI powder in water, fi l-
tering and aliquoting in tubes. If not used, they can be stored
at −20 °C. Note that PI can also be used to detect dead cells:
PI does not go through an intact plasma membranes and binds
to the cell wall, but in a damaged cells, it enters into the cell
and binds to DNA [
25
].
μ
3
Methods
3.1 Accessing
the Meristem
1. In Arabidopsis, when plants on soil start to bolt (i.e., when the
infl orescence stem is about 2 cm above the rosette leaves), the
stem is cut out and placed vertically in box containing 1 % agar
MS medium (
see
Note 2
). Note that as the size of the
Arabidopsis infl orescence meristem is decreasing as stem elon-
gates, we recommend using short stems, where the apical mer-
istem is easier to dissect and handle. Using a pair of forceps
(that were previously sharpened as much as possible on a
stone), fl oral buds are removed from the most aged to the
youngest, by clipping them out. To remove the younger buds,
a good binocular is absolutely necessary. In the end, a glossy
dark green dome surrounded by light green young organs
should be visible—it corresponds to the infl orescence meri-
stem (Fig.
1
;
see
Note 3
). Organs that are not obstructing the
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