Biology Reference
In-Depth Information
defi ned polysaccharide features. These probes may be directly
coupled to a fl uorescent tag or used in conjunction with a tagged
secondary antibody with specifi city to the primary probe. Whole-
mount labelling is a relatively quick and useful technique to assess
cell wall composition at unadhered surfaces, whereas sectioning of
embedded material enables mapping of cell wall polymer microdo-
mains in internal cells and tissues without loss of context. By using
immunocytochemical analyses in combination with chemical and
enzymatic cell wall disassembly, one can investigate whether spe-
cifi c cell wall polymers have the capacity to block probe access to,
or “mask,” underlying cell wall architectures, thereby giving insight
into possible polymer interactions in muro [ 3 , 4 ]. With the increas-
ing number of probes now available making analyses costly both in
terms of time and money, strategies for choosing suitable probes
and treatments are discussed. Here we focus on resin embedding
and sectioning of hard tissues; wax embedding of soft tissues is
discussed elsewhere [ 5 ].
2
Materials
Prepare all solutions using deionized water and analytical grade
reagents. Prepare and store all reagents at 4 °C (unless indicated
otherwise).
2.1 Molecular
Probes
1. Large panels of MAB probes with specifi cities to plant cell wall
polysaccharides and proteoglycans are available from the fol-
lowing suppliers: Biosupplies ( http://www.biosupplies.com.
au ), Carbosource Services ( http://www.carbosource.net ) and
PlantProbes ( http://www.plantprobes.net ). Biosupplies and
Carbosource MABs are raised using mouse hybridoma tech-
nology, and thus the probes require anti-mouse secondary
reagents; PlantProbes provides mostly rat MABs and thus
require anti-rat secondary reagents. Secondary reagents with a
range of tags depending on the particular application are avail-
able at Sigma-Aldrich ( http://www.sigmaaldrich.com ). The
available panel of probes is now considerable (see supplier
websites for full details), thus one must choose carefully
depending on the system under study ( see Note 1 ).
2. Recombinant CBMs are derived from microbial glycosyl hydro-
lases and engineered with a polyhistidine (His) tag to allow
detection with anti-His secondary reagents. They may also be
engineered with a directly coupled fl uorescent protein, such as
GFP, allowing direct visualization of probe binding by epifl uo-
rescence microscopy. Although not yet widely used, some are
available commercially ( see http://www.plantprobes.net ) .
Search WWH ::




Custom Search