Resin Embedding, Sectioning, and Immunocytochemical Analyses of Plant Cell Walls in Hard Tissues - Plant Cell Morphogenesis: Methods and Protocols - page 49

2. The most important step in preparing material for sectioning
and microscopic analyses is the killing and preservation of cells
and tissues. Chemical fi xation of tissues can produce structural
artifacts (swelling and shrinkage) and so it is important to be
aware of this prior to microscope analyses of sections. Fixatives
work by cross-linking proteins, lipids, and nucleic acids. They
do not cross-link polysaccharides and so some polysaccharides
may be lost during sample preparation. If one suspects this to
be the case, it may be useful to analyze a fresh hand-cut section
of the sample or by performing a tissue print [ 9 ]. GA forms a
dense, extensive cross-linked matrix and is therefore better
than PFA at preserving the fi ne structure of cells. Furthermore,
GA cross-linking reactions reach end point much quicker than
PFA. However, GA has a slower infi ltration rate than PFA and
so more time should be allowed for sample fi xation. The
extensive cross-linked matrix of GA can impact on antibody
reactions, for this reason it may be preferable to use GA/PFA
in combination to take advantage of the best qualities from
both aldehydes. Common compositions are 2 % (w/v)
PFA + 1 % (w/v) GA and 2.5 % (w/v) PFA + 0.25 % (w/v) GA;
the latter ensures tissue stability while retaining probe acces-
sibility. Both GA and PFA fi xation of tissue can generate fl uo-
rescent compounds, although the effect is more pronounced
with GA. In general this is not a problem when analyzing
resin-embedded samples. However, sample autofl uorescence
can be quenched by post-staining sections with 0.1 % (w/v)
Toluidine Blue O in 0.1 M sodium phosphate buffer, pH 5.5
after immunolabelling.
3. Pectic HG has been shown to mask XG and heteromannan
polysaccharides. Pectic HG may be removed from resin sec-
tions by treatment with either pectate lyase or polygalacturo-
nase. Both enzymes are active on unsubstituted HG polymers;
therefore section pretreatment with alkali to remove esters
may optimize enzyme action and subsequent HG removal.
Masking of cell wall polysaccharides may be a general feature
of cell wall architecture and so pretreatment of sections with a
range of glycosyl hydrolases against the constituent polymer
classes may reveal subtleties in wall composition and architec-
tures and give insight into polymer associations in muro .
4. For larger samples or in cases where sample processing pro-
duces poor quality blocks with insuffi cient infi ltration of resin,
incubation of the sample at each dehydration and infi ltration
step may be extended to 24 h.
5. Although large gelatin capsules are available from Agar
Scientifi c that enable embedding of materials with ~10 mm
diameter, it should be noted that longer incubation times
must be employed during sample preparation to ensure proper