Essential Methods of Plant Sample Preparation for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 4

Fig. 1 Hand sectioning can be done either with bare hand or with hand microtome ( a ), a simple device with a
micrometric screw, which allows stepwise adjustment of section thickness. Scale on the microtome is nor-
mally graduated in 10 m increments. Object fi xed into the central clamp is cut with a straight razor or other

suitable blade. To slide the blade along the glass plate smoothly, the blade should have a fl at grind on the glass
touching side. Press the straight blade slightly on the glass plate of hand microtome with thumb and
smoothly slide along to cut the sections ( b ). Notice the position of hands during freehand sectioning ( c ). Section should
be kept permanently moistened with drop of water or buffer. To strop the straight blade ( d ), place the blade fl at
on the strop and draw it spine fi rst along the strop so that the whole length of edge is treated. Rotate the
blade over its spine, so the edge moves away from the strop and draw the blade back. Repeat as long as
necessary.( e ) Simple sections staining holders made of Eppendorf vials, tubing ring, and fi ne mesh

μ

toxic xylene. In our laboratory we generally use n-butanol.
t-Butanol is more effi cient solvent of paraffi n and more potent to
be used for infi ltration. However, high melting point (Tm 25 °C,
frequently solid at lab temperature) and higher price of t-butanol
make n-butanol the easier option.
There are various protocols for the paraffi n infi ltration and
embedding, which might differ in tissue damage and time con-
sumption. Dehydration and paraffi n infi ltration are steps which
usually induce most of the tissue shrinkage. To minimize volume
changes caused by intense solvent exchange, gradual series of solu-
tions with decreasing water content are commonly employed.
Various solvents were proposed for use in dehydration (isopropa-
nol, acetone, methyl cellosolve, etc.) and paraffi n infi ltration (chlo-
roform, xylene, n-propanol, n- or t-butanol; reviewed in ref. [ 3 ]).
Ethanol-butanol dehydration series [ 29 , 30 ] became a method of