Biomedical Engineering Reference
In-Depth Information
9. Rinse in deionized water for 5 min, twice.
10. Incubate cells with monovalent conjugated with NANOGOLD
Fab fragments of antibody against IgG of the species where the
second 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.
11. Rinse with PBS-BSA (3 × 1 min) and then PBS (3 × 1 min).
12. Rinse with deionized water (2 × 1 min).
13. Optional (may reduce background): Rinse with 0.02 M sodium
citrate buffer, pH 7.0 (3 × 5 min).
14. Prepare HQ SILVER™. Incubate cells with HQ SILVER for
time, which gives 20 nm gold-silver particles.
15. Rinse with deionized water (2 × 1 min).
16. Fix with 1 % glutaraldehyde in 0.2 M HEPES buffer (pH, 7.2)
for 10 min.
17. Wash in the same 0.2 M HEPES buffer.
18. To avoid dissolution of silver particles in OsO4, fi x cells with
1 % OsO4 intermitting with thiocarbohydrazide (
see
below)
using the shortened scheme and embed in Epon (
see
Note 3
).
3.5 Immunolabeling
for EM with Antibodies
Conjugated with HRP
1. Wash the fi xed cells for 3 × 2 min with PBS.
2. Incubate the cells with the blocking solution for 30 min and
then with the primary antibodies diluted in blocking solution,
overnight.
3. Wash the cells for 6 × 2 min with PBS.
4. Incubate the cells with the HRP-conjugated Fab fragments of
the secondary antibody for 2 h.
5. Wash the cells again for 6 × 2 min with PBS.
6. Fix the cells with 1 % glutaraldehyde in 0.2 M HEPES buffer
for 5 min.
7. Wash the cells 3 × 5 min with PBS, and then incubate them
with the DAB solution. A successful peroxidase reaction results
in a slightly brown staining of the cells.
8. Finally, wash the cells for 3 × 2 min with PBS and start
embedding procedure (
see
below).
Protocol 6: Enhancement of Sample Contrast, Sample
Locating, and Embedding
1. Wash cells three times with 0.2 M cacodylate buffer (pH 6.9).
2. Treat cells with the reduced osmium for 1 h on ice in the dark-
ness (if one uses silver enhancement during the next steps, the
incubation time should be 5 min).
