Biology Reference
In-Depth Information
3. The aim of all immunolabeling procedures is to obtain a maxi-
mum specifi c signal (refl ecting the distribution of the antigen)
with a minimum of nonspecifi c background. In case of a low
signal to noise ratio, fi rst try to fi nd the optimum concentrations
of primary and secondary antibodies (compare Notes 9 and
10 ). Then the concentration and composition of blocking buf-
fer should be varied in order to reduce nonspecifi c binding of
antibodies or colloidal gold to the sample (compare Notes 5 - 8 ).
Nonspecifi c labeling is mostly a problem with primary antibod-
ies. They often stick to cell walls, chloroplasts, and starch gran-
ules even in well-fi xed and embedded cells or tissues as well as to
the Formvar fi lm.
4. Fast stirring will produce excessive BSA foam.
5. If sample contains positively charged components which
unspecifi cally attract negatively charged gold particles, the
ionic concentration of the Tris-HCl buffer might be too low.
In that case the use of TBS or PBS is suggested.
6. BSA saturates nonspecifi c binding sites and neutralizes posi-
tively charged samples. The BSA should be free of IgGs.
Acetylated BSA (available from Aurion) has a much higher
binding affi nity than non-acetylated BSA and more effectively
blocks polycationic sites. The concentration of BSA should
vary between 0.1 and 2.5 %. Instead of or additionally to BSA,
pre- or nonimmune serum from the host species of the sec-
ondary antibody can be used up to 5 %.
7. Tween facilitates wetting of the sections and prevents hydro-
phobic interactions between gold particles and sample; the
concentration should vary between 0.1 and 1 %.
8. Glycine masks free aldehyde groups introduced by chemical
fi xation; the concentration should vary between 10 and
50 mM. Free aldehyde groups can also be quenched by wash-
ing with 10-100 mM NH 4 Cl, pH 7, or by 0.1 % NaBH 4 .
9. The appropriate concentration of the antibodies is crucial for
successful immunolabeling. When a new primary antibody is
applied, a dilution series between 1:1 and 1:5,000 should be
tested. In general, EM immunolabeling requires an up to 10×
higher concentration of primary antibody than used for
Western blotting because of the lower number of binding
sites. Ideally, the primary antibody should be diluted so that
nonspecifi c binding is reduced below the level of detection.
10. At the optimum concentration no gold conjugates should be
present on sections in the absence of the primary antibody
(compare Note 13 ).
11. Tweezer points should always be protected with a cap or fi xed
by wire wrapped around the levers. Damaged tweezers can be
deburred by pulling a fi ngernail fi le or a double-sided sandpa-
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