Laser Microdissection of Plant Cells - Plant Cell Morphogenesis: Methods and Protocols - page 245

4. The plant tissue is embedded in “optimal cutting temperature”
compound (OCT) or carboxymethylcellulose (CMC; [ 17 ])
and frozen in liquid nitrogen.

5. Specimens are stored at −80 °C until sectioning.

5.1 Sectioning of
Paraffi n: Embedded
Samples

1. Sectioning of the paraffi n-embedded specimens is performed
with a microtome. Typically 10-15 m sections are generated.

2. The sections are transferred to specifi c membrane or plain
microscopic slides and are dried at 42 °C O/N ( see Note 5 ).
3. Sections are stored at 4 °C under dehydrating conditions [ 7 ,
14 , 19 ].
4. Prior to laser microdissection the specimens have to be depar-
affi nized two times with xylene for 5 min [ 13 , 18 ].
5. Then samples are air-dried.

μ

5.2 Sectioning
of OCT-Embedded
Samples

1. OCT-embedded tissues are sectioned with a cryo-microtome
and mounted to membrane slides or on plain microscopic
slides.
2. To remove the embedding medium, the sections on the slides
are treated with 70 % ethanol at −20 °C for 1 min.
3. Sections are dehydrated with ethanol (95, 100 %) at room
temperature for 1 min and subsequently with xylene for 2 min
[ 2 , 3 , 8 ].
4. A modifi ed protocol to remove OCT is described in ref. [ 24 ].

6 Notes

1. The yield of RNA in plant cells and tissues can vary between
two- and threefold which requires optimized LMD protocols
adapted to each tissue and cell type under analysis [ 18 ].
2. Two precipitative fi xation methods (Farmer's fi xative and
methacarn) combined with classical paraffi n embedding and
microwave paraffi n embedding were tested for Arabidopsis
thaliana leaves. After fi xation, cells were sampled and the iso-
lated RNA was tested by RT-PCR. The results did not show
any signifi cant differences between the applied approaches
[ 18 ]. However, the amplifi cation of transcripts did not provide
any information on the quality of the underlying RNA [ 10 ].

3. Since LMD yields typically only minute amounts of biomole-
cules, it is recommended that their quality is assessed for
instance by Bioanalyzer analyses prior to subsequent down-
stream analyses [ 10 , 16 , 17 ].