Light Microscopy Protocol
Immuno Incubation
Procedure
using Ultra Small
Immunogold Reagents
(subnanometer gold particles)
A rocking table is recommended for facilitated penetration and reagent exchange.
1. To inactivate residual aldehyde groups present after aldehyde fixation specimens are incubated with 0.1% NaBH4 in PBS buffer for 15-30 minutes.
2. Wash with PBS for 3x10 minutes
3. When working with tissue slices or compact material a pre-treatment with detergent is required to provide acces to internal antigens. In practice: permeabilize with 0.05% Triton-X-100 in PBS for 30 min.
4. Transfer the specimens into matching AURION BLOCKING SOLUTION for 30 minutes up to 1 hour.
5. The specimens are washed with INCUBATION SOLUTION for 2 x 10 minutes.
6. Incubate with a dilution of specific primary antibody,
preferably affinity-purified, 1-5 µg/ml, or a high dilution of a high
titre antiserum, made up in INCUBATION SOLUTION for at least 1
hour.
Antibody concentration and incubation time may have to be adapted
according to the specific characteristics of the primary antibody.
Longer incubation times are usually required to warrant full penetration to antigens. In those cases the procedure should be carried out at 4°C .
7. The specimens are washed with INCUBATION SOLUTION for 6 x 10 minutes.
For
Streptavidin reagents in a three step labeling set-up only:
Incubate with
the biotinylated secondary antibody according to step 5, rinse according to
step 6 and proceed with step 7.
8. The specimens are transferred into aliquots of the appropriate gold conjugate reagent, diluted 1/50-1/200 in INCUBATION SOLUTION for 30 minutes to 2 hours. It is recommended to test a series of dilutions and incubation times for each new localisation study.
Again, longer incubation times are usually required to warrant full penetration to antigens. In those cases the procedure should be carried out at 4°C .
9. The specimens are washed with INCUBATION SOLUTION for 6 x 10 minutes.
10. Wash twice with PBS for 10 minutes each, postfix in 2% glutaraldehyde in PBS for 15 minutes and finally wash with distilled water for 4x10 minutes.
11. Proceed with Silver Enhancement using AURION R-Gent SE-LM as described in the adjacent section.
