Biomedical Engineering Reference
In-Depth Information
4. Dilute the NANOGOLD-conjugated Fab fragments of the
secondary antibodies x50 times in the blocking solution, and
add this to the cells; incubate for 2 h.
5. Wash the cells again for 6 × 2 min with 0.2 M HEPES
(PH 7.2-7.3).
6. Fix the cells with 1 % glutaraldehyde in 0.2 M HEPES buffer
(pH 7.2) for 5 min.
7. Wash the cells for 3 × 5 min with 0.2 M HEPES (PH 7.2-7.3)
and then for 3 × 5 min in distilled water.
8. Incubate the cells with the gold enhancement mixture for
6-10 min according to instruction of the manufacturer. The
cells will become violet-gray in color if the gold enhancement
is successful.
9. Wash cells for 3 × 5 min with distilled water (
see
Note 2
).
3.4 Silver
Enhancement for
Double Labeling
1. Incubate cells with 1 % bovine serum albumin in PBS at pH 7.4
(PBS-BSA) for 5 min; this manipulation blocks any nonspe-
cifi c protein-binding sites and minimizes nonspecifi c antibody
binding.
2. Incubate with mixture of primary antibodies obtained from
different species and diluted at usual working concentration in
PBS-BSA (30 min-1 h or usual time)
3. Rinse with PBS-BSA (3 × 1 min).
4. Incubate cells with monovalent conjugated with NANOGOLD
Fab fragments of antibody against IgG of the species where the
fi rst primary antibody was developed (NANOGOLD
®
reagent
should be diluted 1/40-1/200 in PBS-BSA with 1 % normal
serum from the same species as the NANOGOLD
®
reagent)
for 30 min at room temperature.
5. Rinse with PBS-BSA (3 × 1 min) and then PBS (3 × 1 min).
6. Rinse with deionized water (2 × 1 min).
7. Optional (may reduce background): Rinse with 0.02 M sodium
citrate buffer, pH 7.0 (3 × 5 mins).
8. Prepare HQ SILVER™ using equal amounts of the three com-
ponents. Dispense initiator (A) into a clean tube or dish, add
moderator (B), and mix thoroughly, then add activator (C),
and mix thoroughly again to prepare the reagent. Develop
specimen for 30 s-4 min. More or less time can be used to
control particle size and intensity of signal. A series of different
development times (i.e., 30, 60, 120 s) should be tried to fi nd
the optimum time for your experiment. Select the time, which
gives the size of gold-silver particles of 20 nm. Incubate cells
with HQ SILVER for time, which gives 20 nm gold-silver
particles.
