Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues - Plant Cell Morphogenesis: Methods and Protocols - page 45

2. For hard materials such as plant seeds, it is necessary to create
a hole in the sample using a needle to allow solutions to pen-
etrate to the center of the sample ( see Note 4 ).
3. For large, stiff materials, such as stems, the best results will be
obtained by hand sectioning the material into blocks or slices
no larger than 5 mm thick prior to fi xation and embedding
( see Note 5 ).
4. Use 4 % (w/v) PFA in PEM buffer for epifl uorescence micros-
copy of semi-thin (0.5 m) resin sections. Use 2.5 % (w/v)

GA in 0.1 M sodium cacodylate buffer for electron micros-
copy analyses of ultrathin (~80 nm) resin sections.
5. Small samples are fi xed under vacuum (to expel air) for at least
1 h at room temperature. For larger samples it may be necessary
to increase the incubation time, but for no more than
overnight. Incubation of samples in fi xative for longer periods
increases sample autofl uorescence and can make the sample
brittle. After fi xation samples should be transferred to PEM or
PBS buffer and stored at 4 °C until use.

μ

3.2 Resin Embedding

1. Wash fi xed material in PEM or PBS buffer three times, each
for 10 min (or overnight at 4 °C).
2. Dehydrate by incubation in an ascending ethanol series—10,
20, 30, 50, 70, 90 %, and two times 100 % (v/v)—with 30 min
incubation at 4 °C for each solution. Ensure a suffi ciently large
volume of liquid is used for the sample and place samples in
tubes on a rotator.
3. Infi ltrate with resin by incubation in an ascending resin series
of 10, 20, 30, 50, 70, and 90 % (v/v) resin in ethanol with a
1 h incubation at 4 °C for each solution. Finally transfer sam-
ples to 100 % (v/v) resin and incubate overnight, then 8 h,
then overnight.
4. Transfer samples to gelatin capsules containing fresh resin and
ensure appropriate orientation of plant material (capsules may
be examined under a dissecting microscope if samples are very
small). Fill to the top with resin and seal to exclude air. It is
useful to prepare several capsules containing the same sample
as some may have poor orientation and so may not be suitable
for sectioning.
5. Allow polymerization of resin either at 60 °C for 24-48 h,
37 °C for 5 days, or by the action of UV light at −20 °C.

3.3 Sectioning
of Resin-Embedded
Material

These instructions relate to the use of a Reichert-Jung Ultracut E
Ultramicrotome (Reichert, Vienna, Austria).

1. Using an in-house prepared glass knife, trim the resin block
until your sample is visible at the cut face of the block.