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 4×5 minutes.
9. Proceed with Silver Enhancement 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. Grids are floated on drops of enhancement mixture. 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 grids are washed extensively on drops of distilled water (at least 3×10 minutes). A postfixation with photographic fixer is not required.
4. Grids are contrasted according to standard procedures.