Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 10

2. Wash out fi xative with 15 % (aq. v/v) glycerol containing 2 %
(v/v) of dimethyl sulfoxide and leave for 30 min.
3. Replace the solution with 30 % glycerol containing 2 % of
DMSO and leave for 30 min.
4. Transfer into 50 % glycerol and leave for 30 min.
5. Replace solution with 65 % glycerol and leave for 30 min.
6. Mount the objects into NaI clearing solution and apply cover
slip. Let the objects to clear up. In most cases 24 h is suffi cient,
for more voluminous objects time should be prolonged.
7. Preparations can be saved for weeks at 4 °C.

3.3 Freehand
Sectioning

Good quality double-sided razor blade is indispensable to success-
fully cut objects in bare hands. Quality of the blade makes strong
limitation to the quality and attainable thickness of the sections.
Longitudinal splitting of the blade is rather convenient practice.
Besides better handling it is easier to control which side is still fresh
and having good edge.
1. Grip the sample as indicated in Fig. 1c . For larger axially sym-
metric objects, it does not make much sense to intent to cut
complete sections. More convenient is to get partial but thin-
ner sections. If the object is too thin to be griped, it should be
supported with some moist material and cut within such a
material. We prefer soft elder pith soaked with appropriate buf-
fer of water.
2. Wet the blade and cut the section holding the object more/
less vertically (Fig. 1c ). It is convenient to use fi ne brush to
collect and manipulate sections. Always keep the sections in
solution as drying of the tissue is destructive and rapid at lab
temperature.
3. Holders made of Eppendorf vials (with conical part cut off),
a ring of tubing (inside diameter 10 mm), and fi ne mesh
(Fig. 1e ) might be used for convenient handling of sections
( see also ref. [ 42 ]).

3.4 Hand-Microtome
Sectioning

Razor blade should be kept very sharp during the sectioning (for
maintenance see Note 10 and Fig. 1d ).

1. Preparation of the sample is identical to freehand sectioning
(see above).
2. Clamp the specimen in the central position of microtome so
that it extends over the fl at glass plate. If necessary, use
supporting material (e.g., water-soaked elder pith or carrot
sticks) to fi x small specimens in appropriate position similarly
to freehand sectioning (Fig. 1b ).
3. Carefully place the fl at side of straight blade on the glass plate
and cut the object to align it with the plate (sectioning plane).