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,
http://cell.ccrc.uga.edu/~mao/wallmab/Antibodies/antib.
htm
). Some of them were used in our lab in protocol similar to
protocol presented bellow for callose.
1.3 Staining of Cell
Wall Lipids and Lignin
Presence of lipidic compounds in cell wall is frequently connected
with formation of cell layers with modifi ed permeability of apo-
plast. Two principal insoluble cell wall lipids were historically dis-
tinguished by their position. Suberin is located in internal and
secondary dermal tissues, while cutin constitutes cuticular part of
epidermis on the surface of plant organs [
20
]. Considerable varia-
tion in monomeric composition of suberin and proportion of aro-
matic and lipidic domain were reported in between species and
during development [
21
,
22
]. There are several staining proce-
dures used for detection of lipidic compounds in cell walls. Lipidic
Sudan dyes (Sudan III, Sudan IV, Sudan Black B) are traditionally
utilized in alcoholic solutions. Polyethylene glycol/glycerol-based
staining solution of Sudan red 7B introduced by Brundrett [
23
]
proved to be far more effi cient and is the method of choice. Lipidic
dyes partition from the slightly polar dyeing solution into the
lipidic compartments of the tissue. It should be emphasized that
intensity of staining depends highly on lipidic nature of cell wall
material (quantity as well as molecular context of derivatives of
fatty acid). Therefore sensitivity of the detection should be consid-
ered during interpretation, and nonspecifi c precipitation should be
avoided. Improvement of sensitivity was reported due to use of
lipidic fl uorochrome Fluorol yellow [
23
]. However, background
staining and autofl uorescence can be sometimes diffi cult to distin-
guish from specifi c Fluorol yellow signal. That is why for some
objects (e.g., maize roots) Sudan red 7B is preferred in our hands.
Commonly we use fresh sections after aldehyde or no fi xation.
Several pitfalls are known (see method description below). Modifi ed
method of Fluorol yellow staining combined with lactic acid clear-
ing of object was published by Lux [
24
]. Finally a very old tech-
nique of concentrated sulphuric acid digestion of cell wall material
might also provide valuable information as only suberin and cutin
impregnated material should resist it [
25
,
26
].
Berberine-toluidine blue staining procedure was introduced
by Brundrett et al. [
27
] to detect material of Casparian bands,
suberin lamellae, and lignifi ed tissue. It is a very frequently used
staining based on acidophilic nature of berberine, which stains aro-
matic domains of lignifi ed and suberised cell walls. The staining of
berberine is combined with counter stain of toluidine blue (alter-
natively aniline blue, Evans blue, or Crystal violet) to quench
background fl uorescence. Selectivity of such a quenching is most
likely related to physical properties of the cell wall (decreased acces-
sibility of the material, e.g., due to suberinization). In fact counter-
staining itself provides valuable information and combination with
other acidophilic fl uorochromes (e.g., acridine orange) or observa-
tion of autofl uorescence of suberised cell walls is possible.
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