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FIGURE 1. Specificity of
the antibodies for their respective
antigens. Multiwell
plates were coated
with 50 ng of the protein
designated as antigen, in
50 #1 of buffered saline.
All remaining, nonspecific
binding sites for protein
were then saturated with
8SA. Solutions in buffered
saline of antivinculin (48
ng/ml), anti-a-actinin (144
ng/ml), antifilamin (208
ng/ml), and antimyosin
(160 ng/ml) were introduced
in 50-ttl aliquots and
incubated for 1.5 h at 37 °.
Unbound antibody was
washed out, and goat-antirabbit
IgG coupled to alkaline
phosphatase (270
ng/ml) was introduced and
incubated, as described.
Bound antibody-linked alkaline
phosphatase was assayed using p-nitrophenyl phosphate (0.6
mg/ml in diethanolamine buffer, pH 9.8). More details are given in
reference 9. F, filamin; V, vinculin; A, a-actinin; M, myosin; R,
tropomyosin; G, gizzard actin; S, skeletal muscle actin; T, tubulin;
and O, blank. Similar results were obtained with sixfold higher
antibody concentrations. The results show that each antibody is
highly specific for its appropriate antigen.
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FIGURE 2. Antibodies against
vinculin, e~-actinin, and filamin
stain the neuromuscular junction
of rat diaphragm. Frozen
transverse sections were cut
through junctional regions of
normal, adult rat diaphragm.
Sections were reacted with (a
and b), antivinculin (12/~g/ml);
(c and d), anti-a-actinin (36
#g/ml); (e and f) antifilamin (34
ttg/ml). Counterstaining was
with R-a-BuTx and either
fGAR in a-d, or with biotinylated
GAR and fluoresceinated
avidin, in e-f. After staining,
samples were mounted in
Elvanol and observed under
epi-illumination to visualize
fluorescein (b, d, f) and tetramethylrhodamine
(a, c, e). The
results show that the three antibodies
react with junctional
regions, labeled with R-cz-
Bul-x. Bar in t', 25/~m.
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FIGURE 3. Specificity of antivincuJin
staining of the neuromuscular
junction. The procedure
given in the legend to Fig. 1 was
followed, but only antivinculin
was used. b, d, f, h, and j show
fluorescein fluorescence obtained
after staining with fGAR. a,
c, e, g, and i show staining of the
AChR in the same sections with
R-a-BuTx. the antibodies used
were (a and b) antivinculin (12 pg/
ml); (c and d) nonspecific IgG
from antivinculin antiserum (12
pg/ml), which shows essentially
no staining; (e and t~ antivinculin
(0.38 #g/ml); (g and h) antivinculin
(0.38 #g/ml) preadsorbed with
purified vinculin (7.6 #g/ml) for
15 rain at 22*, which shows significantly
lower staining; (i and j)
antivinculin (12 pg/ml) staining of
a section through the junctional
region of a rat diaphragm that had
been denervated 3 wk before
freezing and sectioning. The results
show that antivinculin staining
of the neuromuscular junction
is specific and that it persists in
long-term denervated muscle.
Note the appearance of intense
extrajunctional stain in denervated
muscle (j). Bars, 20 pro; the
bar in d applies to a-d; in h, to eh;
and in i, to i and i.
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FIGURE 4 Antibody stainingofjunctional
regions from newborn rats.
Frozen sections (6 #m) through diaphragm
obtained from newborn
(<24-h-old) rats are shown. Conditions
are as described in the legend
to Fig. 1. The antibodies are (b), antivinculin;
(d), anti-cz-actinin; (/), antifilamin,
a, c, and e show corresponding
R-a-BuTx staining of the same
sections. The results indicate that
junctional regions of newborn diaphragm
show antivinculin, anti-a-actinin,
and antifilamin staining. Note,
however, that extrajunctional staining
is also apparent (e.g., t3. Bar in h,
10 ~.m.
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FIGURE 5 Staining of neuromuscular junctions of mouse, chicken, and Xenopus. Frozen sections through junctional regions of
mouse diaphragm (a, d, and g), chicken posterior latissimus dorsi (b, e, and h), and Xenopus sartorius (c, f, and i) muscles were
stained with antibodies. Methods were as described in Fig. 1, except that fluorescein-avidin was used only in antifilamin staining
of mouse muscle and staining of chicken muscle used lO-fold lower concentrations of antivinculin and ninefold lower
concentrations of fGAR. R-a-BuTx stained regions (not shown) in all cases coincided with the brightly staining, membraneassociated
structures pictured here. The antibodies used were antivinculin, (a-c); anti-a-actinin, (d-f); antifilamin, (g-i). The
results show that, except for anti-c~-actinin reaction with Xenopus muscle (f), junctional regions of all three species are stained
by the antibodies. Some weak extrajunctional staining is also apparent in a and e; staining of the contractile apparatus is evident
in f, and, more faintly, in d. Bars, 20 pm. The bar in g also applies to a and d, and the bar in i applies to the rest of the panels.
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