Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 9

carboxymethylcellulose (CMC) to increase medium thickness
up to semisolid gel. Add 7-10 g CMC powder on the surface
of the medium, leave to rehydrate overnight, and mix well.
Centrifuge to eliminate air bubbles.
2. Sucrose solutions: 3, 10, and 20 % w/v solutions of sucrose in
0.1 M phosphate buffer (pH 7.4).

3 Methods

3.1 Fixation
of the Samples

1. Cut samples of adequate size to allow rapid access of fi xative to
inner tissues. In general the smaller is the better. On the other
hand, size of structure of interest and/or cell size and investi-
gator desire should be considered during sampling. Use sharp
razor blade to minimize damage in vicinity of cut edge.
2. Submerge samples into adequate volume ( see Note 6 ) of
fi xative solution immediately after excision or cut under suit-
able buffer, water, or cultivation solution to avoid drying. We
found 20 ml scintillation vials to be the convenient vessels for
fi xation.
3. Alcoholic solutions easily fi ll in intercellular spaces due to low
surface tension. If aqueous solutions are used, application of
lower pressure (“vacuum infi ltration”) might be necessary to
substitute air with fi xative solution. Vacuum pump connected
to plastic desiccator via regulator allows for controlled gradual
decrease of pressure within the chamber. The rate of pressure
drop depends on the nature of object and fi xative used and
should be adjusted accordingly. In general decrease should not
cause boiling of the solution, but only slowly escaping stream
of bubbles should be stimulated. Bring the samples slowly
down to minimum pressure of the pump (approx. 5 mBar),
turn off the vacuum line, and let the samples to equilibrate
within the chamber for 10-20 min. Then let air slowly in to fi ll
up the chamber again. The reintroduction of pressure should
be gradual and as gentle as possible to fi ll “vacuum” within the
sample intercellular spaces with solution during this period.
Quick release of pressure difference might cause collapse of the
intercellular spaces.
4. Let the samples to be fi xed for selected period of time ( see
Notes 7 and 8 ).

3.2 Simple Protocol
of Whole-Mount
Samples or Thick
Sections Clearing

Procedure is optimized for Arabidopsis seedlings and might need
minor readjustment for other samples. Multiwell culture plates are
convenient to process larger sets of samples ( see Note 9 ).

1. Fix samples in 4 % formaldehyde buffered to pH 7.2 (25 mM)
overnight.