AURION: Post embedding Ultra Small Gold

Protocol Ultra Small Reagents

aurion Post-embedding Immuno Incubation Procedure
Using AURION Ultra Small Immunogold Reagents
(subnanometer gold particles)

Nickel grids are recommended for compatibility with Silver Enhancement.

1. To inactivate residual aldehyde groups present after aldehyde fixation grids are incubated on 0.05 M Glycine in PBS buffer for 10-20 minutes.

2. Transfer the grids onto drops of the matching AURION BLOCKING SOLUTION for 15 minutes.

3. The grids are washed on drops of INCUBATION SOLUTION for 2 x 5 minutes.

4. The grids are transferred onto drops of 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 30 minutes to 1 hour. Antibody concentration and incubation time may have to be adapted according to the specific characteristics of the primary antibody.

If longer incubation times are required (e.g. with low titre antibody solutions) the procedure should be carried out at 4°C overnight.

5. The grids are washed on drops of INCUBATION SOLUTION for 6 x 5 minutes.

For Streptavidin reagents in a three step labeling set-up only:
Incubate with the biotinylated secondary antibody according to step 4, rinse according to step 5 and proceed with step 6.

6. The grids are transferred to drops 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.

7. The grids are washed on drops of INCUBATION SOLUTION for 6 x 5 minutes.

8. The grids are washed twice on PBS for 5 minutes each, postfixed in 2% glutaraldehyde in PBS for 5 minutes and finally washed on distilled water for 4x5 minutes.

9. Proceed with Silver Enhancement using AURION R-Gent SE-EM as described.

RECOMMENDED FOR EM

Silver enhancement for EM
(when using Ultra Small IGR)

AURION R-Gent SE-EM

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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