Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 25

Even such “anatomical staining” can gain useful results, but inter-
pretation should be very cautious.

Toluidine blue O polychromatic staining is a simple and very useful
oversight staining procedure disseminated in plant microtechnique
by O'Brien et al. [ 1 ]. Besides good overall contrast of most struc-
tures, it also renders information on properties of the stained mate-
rial. That is because the pigment interacts with stained material and
shifts its absorption spectra towards longer wavelengths according
to density of surface polyanions and subsequent dye aggregation
[ 2 ]. Such coloration is referred as a metachromatic [ 3 ]. Therefore
cell walls with low pectin content will stain blue (orthochromatic
color), while pectin rich material will stain purple to pink (meta-
chromatic color). Because lignifi ed/phenolics containing cell walls
present lower concentration of acidic groups, their staining is usu-
ally greenish. Cell wall material can be stained metachromaticaly
above pH 3. Besides, cell wall tannin-containing vacuoles might
stain green to bright blue, DNA-containing nucleus green.
PAS (periodic acid—Schiff 's reagents) reaction is a nonselec-
tive polysaccharide detection procedure [ 4 ]. Periodic acid is a
strong oxidizing agent cleaving vicinal diol linkages of polysaccha-
rides and producing dialdehydes. These are subsequently detected
with Schiff 's reagent or its fl uorescence alternatives [ 5 , 6 ]. However,
there is often background signal of some aldehydes present in the
tissue (e.g., lignin monomers), and some others might be intro-
duced during treatment with aldehyde fi xative, which should be
therefore used with consideration. That is why control sections
without previous periodic acid treatment should be always included
to ascertain about the origin of aldehydes. Optionally the autoch-
thonous aldehydes might be eliminated before periodic acid treat-
ment with borohydride reduction [ 6 ]. The PAS reaction scheme
has been used recently also for staining of whole mount objects in
combination of fl uorescent leucobases of propidium iodide [ 7 ].
Calcofl uor staining might be considered another general pro-
cedure of fl uorescent cell wall accentuation. Calcofl uor (synonyms
are Tinopal, Fluorescent brightener) is nonspecifi c UV excited
fl uorochrome with high affi nity to plant and fungi cell walls [ 8 ]. Its
selectivity is considered to be related to (1

1.1 General Cell Wall
Staining Methods

→ 3), (1 → 4), - β -D-

glucan chains of polysaccharides similarly to Congo red [ 9 ].
Besides procedures of general cell wall detection, there are
methods aimed for specifi c components of cell wall.

1.2 Staining
of Pectins, Callose,
and Hemicelluloses

Alcian Blue is a basic dye, which can be used to rather specifi cally
stain dissociated acidic carboxyl groups of pectins [ 10 ]. Acidic
environment used for staining further narrows spectrum of poten-
tially dissociated (stainable) acidic groups. In fact, there are not
many other compounds that might react with the dye in plant cell
walls under such conditions. Staining mechanism of Alcian Blue is