Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues - Plant Cell Morphogenesis: Methods and Protocols - page 50

buffer exchange and infi ltration of resin. For these reasons it
may be preferable to excise a range of smaller-sized samples
containing features of interest from an organ or tissue, rather
than embedding the entire tissue.
6. A perfect ribbon can only be obtained from a well-trimmed
block. A good quality, well-polymerized acrylic block should
have a glassy surface when cut. It can be diffi cult to cut sec-
tions from wide blocks as this increases the cutting pressure
and may lead to chatter (visible as lines on the cut face of the
block). Compression of the sample may also become an issue
as the face of the block increases in size. If this is the case,
unwanted regions of sample should be trimmed away to leave
a specifi c region of interest. An equivalent block may then be
trimmed in a complementary fashion to give full coverage of
the sample. If one encounters problems with material being
pulled out of the resin during sectioning, microwave infi ltra-
tion and pretreatment with (3-glycidoxypropyl) trimethoxysi-
lane may address this issue [ 10 ].
7. Indirect immunofl uorescence labelling of cell walls is a widely
used technique that can accommodate several antibodies in the
same protocol and also allows assessments of nonspecifi c bind-
ing and sample autofl uorescence. The principles in the immu-
nolabelling procedures are the same for whole-mount labelling
of intact materials and hand-cut sections. Antibody incuba-
tions can be performed in tubes or plates, depending on the
size of the material under study. Direct immunolabelling pro-
cedures, requiring just one step, are rapid, are highly effective,
and may be combined with indirect immunolabelling to enable
dual localization of cell wall epitopes in a single section.
8. The recommended dilution of an antibody is the highest dilu-
tion that results in a strong specifi c signal. Manufacturers of
secondary reagents provide good guidance for dilution fac-
tors. For primary MABs, a 5-10-fold dilution of cell culture
supernatants is often used; however, in some cases, up to a
200-fold dilution can be highly effective in terms of both anal-
yses and costs.
9. Calcofl uor White is used as a counter stain as it binds widely to
β -glycans, including cellulose, and fl uoresces under UV excita-

tion and therefore can indicate all cell walls in sections and is
useful for orientation and identifi cation of immunolabelling in
relation to organ and tissue anatomy. If sample autofl uores-
cence is a problem, equivalent sections of the sample can be
labelled with either MAB and Calcofl uor White or MAB and
Toluidine Blue O to allow visualization of all cell walls and
MAB fl uorescence without the contribution of sample
autofl uorescence.