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 3×10 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 4×10 minutes.
11. Proceed with Silver Enhancement using AURION R-Gent SE-LM
Silver Enhancement for Light Microscopy
1. Once the DEVELOPER and ENHANCER reagents have reached temperature equilibrium, equal parts are mixed immediately before applying the enhancement mixture to the specimens. The specimens should be fully covered by the mixture.
2. Enhancement is done at room temperature (preferably 20°C). Gentle shaking is optional.
3. Typical enhancement times are between 15 and 25 minutes. Auto-nucleation is visible only after 30-40 minutes.
The on-going process may be monitored using an inverted light microscope with dimmed light conditions. In this way heat transfer to the enhancement mixture is kept low.
4. When enhancement is complete (judged by the presence of the brown to black signal developed) the specimens are washed extensively with distilled water (at least 3×5 minutes). A postfixation with photographic fixer is not required.
5. After washing light microscopical specimens may be counterstained according to standard procedures.
6. Short term storage (up to 1 year) can be achieved by mounting in water compatible Glycergel™ (DAKO).
For long term storage it is necessary to dehydrate the silver enhanced specimens and to mount in water-incompatible media such as Pertex (HistoLab, Sweden).