Laser Microdissection of Plant Cells - Plant Cell Morphogenesis: Methods and Protocols - page 240

The isolation of specifi c cells is straightforward for only a few very
exposed plant cell types such as root hairs [ 4 ] and leaf trichomes
[ 5 ]. However, most cell types are interconnected with other cells
deep inside a complex tissue. Specifi c cell types expressing fl uores-
cent marker genes can be separated by fl uorescent-activated cell
sorting (FACS) after protoplasting [ 6 ]. If no such marker lines are
available, cells that can be microscopically distinguished from
other cell types can be mechanically separated from their cellular
context by laser microdissection (LMD). For laser microdissection
in general, two types of techniques have been developed to sepa-
rate specifi c cell types from the surrounding tissue. In the fi rst
“touch-free” approach, cells of interest are cut out and transported
away from their tissue context either by gravity or by catapulting
them away e.g., Veritas Laser Microdissection LCC1704
(Arcturus), PALM MicroBeam (Zeiss), or AS LDM (Leica). In an
alternative approach, cells of interest are melted to a plastic
membrane or are brought in contact with a sticky surface and are
subsequently removed mechanically from the remaining tissue
e.g., PixCell II LCM (Arcturus).
The plant kingdom consists of a wide variety of diverse plant
species with a considerable number of functionally diverse cell
types ( see Note 1 ). Laser microdissection remains challenging
because there is no general protocol available that is suitable for all
plant species and cell types. Hence, optimal protocols have to be
established for each application. The quality of tissue sections and
the integrity of biomolecules such as RNA or proteins in these
samples is the major prerequisite for successful LMD analyses. The
initial decision on the fi xation and embedding method is already
decisive for the success of the experiment. In general, two types of
fi xation are applied: precipitative and cross-linking fi xation.
Typically, precipitative fi xation (Farmer's fi xative, see Note 2 ) yields
higher RNA concentrations ( see Note 3 ) than cross-linking fi xation
(formaldehyde-acetic acid-ethanol; [ 7 , 8 ]). In most experiments,
either frozen plant tissues (embedded in OCT (optimal cutting
temperature compound) or CMC (carboxymethylcellulose)) or par-
affi n-embedded plant tissues are sectioned. The morphology of
sections is often better preserved in paraffi n-embedded tissues [ 9 ].
However, in many instances frozen tissues provide higher yield and
quality of RNA or other biomolecules under analysis ( see Note 4 ).
Hence, in each experiment a balance between conservation of mor-
phology and preservation of biomolecules needs to be achieved.
Another crucial step that determines the quality and integrity
of nucleic acids is the mounting of paraffi n sections to the slide.
RNA quality can be affected during the stretching of paraffi n rib-
bons on water and the drying process afterwards ([ 10 ]; see Note 5 ).
Thus far, only a small number of LMD studies combined with
proteomics experiments have been performed. Farmer's fi xative
and embedding of the samples in OCT to perform cryo-sectioning