Selected Simple Methods of Plant Cell Wall Histochemistry and Staining for Light Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 26

rather similar to ruthenium red [ 11 ]. Ruthenium red is a hexava-
lent cation, which binds to variety of polyanions; that is why this
classical reaction with pectin should be considered typical rather
than specifi c. Ruthenium red also has its traditional use in electron
microscopy (e.g., [ 12 ]).
Specifi city of the staining can be further verifi ed after pointed
carboxyl blockage via methylation [ 4 ]. Blockage of acidic carboxyl
should also abolish most of toluidine blue metachromasy discussed
above. Pectins can be often (depending on the linkages within the
cell wall context) extracted with hot aqueous solutions, Ca 2+ che-
lating agents, and weak alkali solutions. Therefore such treatment
should be avoided prior to pectin staining with any of the methods
described. On the other hand extracting agents and their sequences
might be used in connection with detection methods to further
specify or confi rm composition of extracellular material according
to specifi c extractability (e.g., [ 13 , 14 ]).
Callose is highly dynamic polymer (e.g., [ 15 ]). Its presence in
tissues might be easily induced, for example, with chemical fi xation
of samples. Callose deposition is one of rather fast responses to
stress or plant cell injury. Also aldehyde fi xation (in fact it is a kind
of chemical injury) induces deposition of callose into the plasmo-
desmata containing pit fi elds in order of minutes. That is why usage
of cold methanol fi xation or callose synthase inhibitors proved to
be convenient to approach in vivo presence of callose. There are
two most common ways of callose detection in tissues.
The most frequently used is staining with aniline blue [ 16 , 17 ],
respective its common impurity—the UV excited fl uorochrome
Sirofl uor. Because the content of Sirofl uor in the raw dye is variable
according to brand and batch, it is reasonable to test your dye stock
with known material fi rst or use purifi ed (and far more expensive)
fl uorochrome, which form highly fl uorescent complexes with (1-3)
β -D-glucans [ 18 ]. Advantage of purifi ed fl uorochrome might be

seen also in the extended staining pH range from 3 to 10, while
aniline blue staining should be at higher pH [ 18 ]. There are several
reports on compromised specifi city of the reaction and possible
interaction with other polymers [ 19 ]. Control unstained section are
convenient to ensure about nature of the fl uorescence emission.
Besides the fl uorescent staining, bright fi eld visualization of callose
with reasonable specifi city might be gained with Resorcin blue [ 17 ].
Since the antibodies are commercially available (e.g., Biosupplies,
Australia), callose immunolocalization provides easily accessible
highly consistent, sensitive, and specifi c detection alternative.
As far as we are aware there is no reliable and specifi c
histochemical test for identifi cation of hemicelluloses. This gap can
be effi ciently fi lled with the use of specifi c antibodies, which allow
for precise distinction of various cell wall components (including
pectins, hemicelluloses, and proteins). There are several sources of
the antibodies currently available (e.g., http://www.plantprobes.