Protocol Pre-embedding

aurion Pre-embedding immuno incubation procedure
using Ultra Small Immunogold Reagents
(subnanometer gold particles)

A rocking table is recommended for facilitated penetration and reagent exchange. Compared to post-embedding procedures longer incubation and wash steps are required.

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-EM as described in the adjacent section.

© Aurion 2007 - 2010

RECOMMENDED FOR EM

Silver enhancement for EM
(when using Ultra Small IGR)
For post-embedding
Glycine to prepare the aldeyde blocking solution
For pre-embedding
Sodium Borohydride to prepare the aldehyde blocking solution
Triton-X-100 for increased accessibility

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