Biology Reference
In-Depth Information
7. Incubate with Calcofl uor White if required (
see
Subheading
3.4
,
step 8
).
8. Mount slides using anti-fade reagent and examine.
1. Block to prevent nonspecifi c binding by fl oating the EM grid
section side down on a droplet (at least 20
3.6 Immunogold
Labelling for Electron
Microscopy
μ
l) of PBS/BSA on
Parafi lm
®
for 30 min.
2. Transfer grid to a droplet of primary antibody diluted in PBS/
BSA. MAB cell culture supernatants should be diluted between
5- and 200-fold.
3. Wash grids by incubation in a minimum of three changes of
PBS, each 10 min.
4. Transfer grids to secondary antibody diluted 20-fold in PBS/
BSA. We routinely use anti-rat IgG coupled to 10 nm gold.
5. Wash as in
step 3
and then extensively in distilled water.
6. Allow the grid to dry and then examine in an electron
microscope.
3.7 Section
Pretreatments Prior
to Immunolabelling
Cell wall polysaccharide epitope masking has been demonstrated in
a number of parenchyma systems [
3
,
4
,
7
,
8
]. Pectic HG is often
methyl esterifi ed, and to effect its most effi cient removal by pectate
lyase or polygalacturonase enzymes, a pretreatment of the section
with a high pH solution is required (
see
Note 3
):
1. Incubate the section with a solution of 0.1 M sodium carbon-
ate (pH 11.4) for 2 h.
2. Wash two times with deionized water, each 10 min.
3. Incubate with pectate lyase (10
μ
g/ml) in CAPS buffer for 2 h
or polygalacturonase (20
μ
g/ml) in sodium acetate buffer
for 2 h.
4. Wash two times with deionized water, each 10 min.
5. Sections are now ready for immunolabelling (
see
Subheadings
3.4
,
3.5
or
3.6
).
6. Do not let sections dry prior to labelling.
4
Notes
1. In the absence of a priori knowledge about the plant tissues of
interest, one may wish to perform a nitrocellulose-based assay
such as a dot blot [
9
] of extracted cell wall material to get an
overview of the polysaccharides present and their relative
abundance. Such an approach will avoid the issues associated
with epitope masking and is a more rapid alternative than sys-
tematically pursuing enzymatic pretreatments of sections when
an immunochemical survey of the tissue is desired.
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