Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 3

procedures combine both attitudes. Hereby we present simple
protocol of gentle tissue clearing with high refractive index solu-
tion, which preserves most of the cellular content. We have intro-
duced into usage sodium iodide solution [ 25 ] as a high refractive
index nontoxic alternative to chloral hydrate, which is a regulated
narcotic in most countries. The procedure is not self-reliant for
highly pigmented and highly optically dense (e.g., secondary
xylem) tissues and should be combined with pigmentation removal
in such a case.

Hand sectioning is fast and easy method of fresh/fi xed specimen
sectioning. While it might seem old fashioned in an equipment-
loaded laboratory, if done with skill, it gains quickly substantial
information on structure and in combination with various detec-
tion techniques also on composition and other parameters of tis-
sue. Freehand sectioning with a razor blade is the simplest option
and should be considered as a basic level laboratory skill. Hand
microtome and straight razor blade (Fig. 1a ) can push the section-
ing further to achieve series of sections of standardized thickness
(

1.3 Hand Sectioning

m is realistic for most tissues). Hand sectioning has no
necessity for infi ltration and embedding. For smaller objects, addi-
tional reinforcement might be necessary to facilitate manipulation
in hand or fi xation in clamp of hand microtome. We commonly use
elder pith (dead parenchymatous tissue), but other material (car-
rot, styrofoam, potato, roll of parafi lm, paraffi n encasing, etc.) or
encasing into agarose block surrounding the object during section-
ing [ 26 , 27 ] might be used. In fact the hand sectioning can pro-
vide sections rather similar to vibratome. Quality of the cutting
edge is the most limiting factor, and high-quality disposable razor
blades (not the single-sided technical ones) or well-maintained
straight razor (requires proper honing and stropping) is crucial for
the sectioning.
The other procedures presented in this selection will involve
specimen infi ltration and embedding with supporting matrix to
form blocks suitable for sectioning. Such embedding allows attain-
ing thinner sections (less than 10

≥ 50 μ

μ m) and routine serial sections.

Paraffi n is the very classical embedding medium introduced into
microtechnique by Klebs [ 28 ]. Paraffi n melts at rather high tem-
peratures (54-60 °C), is strongly hydrophobic, and does not allow
for routine sectioning below approx 3

1.4 Paraffi n
Embedding

m. In spite of these disad-
vantages, it is still the most common embedding medium. Easy
cutting and junction of sections into ribbons allow for straightfor-
ward routine of serial sections. Its high hydrophobicity requires
strict dehydration of the object and use of intermedium (interme-
diate anhydrous paraffi n solvent) to completely saturate tissue with
paraffi n before embedding. Butanol is the most commonly used
intermedium, which substitutes the originally more common and

μ