Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 21

10. Sharpening with fi ne-grained stone (e.g., Japanese whetstone,
grit 8,000) is part of the regular maintenance but does not
replace stropping. The fi nest cutting edge is affected during
sectioning, and frequently it is enough to straighten and polish
the blade by stropping (Fig. 1d ) to recover its sharpness. While
sharpening requires edge-forward movement of the blade on
the stone, it should be carefully drawn spine fi rst to avoid cut-
ting of the strop during stropping. The hard steel of blade is
highly sensitive to corrosion and should be therefore kept away
from acids and stored clean and dry.
11. Timing of individual dehydration steps depends on the size of
the objects and their texture [ 3 ]. For easy objects (e.g., roots
samples with diameter up to 2 mm, leaves 5 × 5 mm segments,
etc.), 3 h in the step should be safe. Small objects up to 1 mm
might require only 15-30 min, while larger objects (minimal
dimension 10-15 mm) might require days per step. Objects of
high tissue density or covered with low permeability cuticle
have signifi cantly impeded exchange of solutions. Timing of
changes should be prolonged accordingly.
12. The dehydration series solutions lose their properties with use,
and contamination with compounds extracted from the sam-
ples takes place. It is recommended to keep record on the
number and type of processed samples and replace solutions
regularly. Precaution is more important for anhydrous (latter)
steps of the series.
13. The embedding can be done using commercially available
molds, cassettes, or origami made of smoothen cardboard
which does not soak up much of the paraffi n (Fig. 2a ). Ceramic
dishes with fl at bottom are also good option. Spray/smear the
ceramic dishes with 50 % glycerol or commercial detergent
before embedding to facilitate latter separation of paraffi n
block from the ceramic surface.
14. Because the knife (as well as disposable blades) is not a simple
wedge but has facets on the edge (Fig. 2c ), the setup of knife
clearance angle should respect the shape. Too low angle will
crush sample; too steep adjustment might cause rolling of sec-
tions and chatter over hard objects. Clearance angle is usually
between 3° and 5° or more acute approx. 10° for thinner sec-
tions and harder objects. Scale for adjustment is normally
marked on the blade holder.
15. Transfer samples into mixture of 20 % sucrose solution and
cryoembedding medium (1:1) and let them infi ltrate over-
night within refrigerator. This step is optional, but introduc-
tion of cryoembedding medium (e.g., OCT) into the object
might further improve quality of sections [ 44 ].