Biology Reference
In-Depth Information
Fig. 1
Epifl uorescence micrographs of tobacco seed endosperm. (
a
) Calcofl uor White labelling indicates all cell
walls. (
b
) A MAB specifi c to
-1-4-linked galactan labels inner cell walls. (
c
) shows merged image.
White lines
indicate positions of middle lamellae. Bar = 6
β
μ
m
2.5 Chemical
and Enzymatic
Pretreatments
1. 0.1 M sodium carbonate pH 11.4. For removal of methyl
groups from polysaccharides.
2. 0.1 M KOH. For the removal of acetyl groups from polysac-
charides [
6
].
3. Pectate lyase (from
Cellvibrio japonicus
) or polygalacturonase
(from
Aspergillus niger
) (Megazyme, Bray, Ireland). For enzy-
matic removal of polysaccharides (
see
Note 3
).
4. 3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS) buffer:
50 mM CAPS, 2 mM CaCl
2
, pH 10 for pectate lyase
treatments.
5. Sodium acetate buffer: 50 mM sodium acetate, pH 4.0 for
polygalacturonase treatments.
3
Methods
Immunofl uorescence microscopy is a sensitive and rapid technique
for the analysis of cell wall architectures in organs and tissues.
Analysis with a 100× oil immersion lens provides an excellent level
of detail and, in a good quality section, one can even resolve indi-
vidual cell walls and middle lamellae using fl uorescent probes and
epifl uorescence microscopy (Fig.
1
). The methods presented here
focus on immunofl uorescence techniques; preparation of samples
for electron microscopy is also discussed and should be employed
when higher-resolution imaging is required to localize cell wall
components in specifi c cell wall domains or organelles. Carry out
all procedures at room temperature unless otherwise specifi ed.
1. The polymerization of LR White resin is inhibited by the pres-
ence of air. It is therefore crucial that samples are well fi xed
and of a small enough size to allow infi ltration of fi xative
solution.
3.1 Plant Material
Preparation and
Fixation
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