Pre-embedding Immuno Incubation Protocol

Recommended Incubation Solution

• PBS (20 mM phosphate buffered saline)
• 0.1 - 0.2% AURION BSA-c
• 15mM NaN3
• check the pH and adjust to 7.4 if necessary

Buffers are either prepared immediately before use or thawed from aliquots stored at -20°C.

Protocol Pre-embedding

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 below.

Silver Enhancement Procedure

Using AURION R-Gent SE-EM (Silver Enhancement for Electron Microscopy)

It is assumed that the Developer has been prepared as prescribed.

1. Prepare the enhancement mixture as follows:

Once the developer and enhancer have reached room temperature, give 20 drops of the enhancer solution into a vial that will contain at least 1.5 ml, e.g. an Eppendorf vial. Make sure to keep the bottle upside down in a vertical position. Add 1 drop  of the developer solution, again making sure that the bottle is kept upside down in a vertical position. Mix well on a vortex.

2. Specimens are floated in enhancement mixture on a rocking table. Enhancement time  is typically between 30 minutes and 1 hour at room temperature (preferably 20°C). The actual enhancement time has to be established empirically and adjusted according to the desired particle growth.

3. When enhancement is complete the specimens are washed extensively with distilled water (at least 3x10 minutes). A postfixation with photographic fixer is not required.

4. Specimens are dehydrated and embedded according to standard procedures.

Silver enhancement and OsO₄ fixation

Silver enhancement may be applied either before or after OsO₄ fixation.  

Enhancement before OsO₄ fixation

The time of enhancement may have to be longer due to potential  removal of silver by OsO₄.

The fixation step is inserted before dehydration.

Enhancement after OsO₄ fixation

The fixation step is inserted before silver enhancement.

Wash extensively after OsO₄ fixation: traces of this strong oxidizing agent will interfere with adequate silver enhancement.

Alternatively enhancement may be done after ultra thin sectioning.