Post-embedding Immuno Incubation Procedure
using AURION Conventional Immunogold Reagents
(6, 10, 15 or 25 nm gold particles)
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 1/20-1/40 in INCUBATION SOLUTION for 30 minutes to 2 hours. It is recommended to test a series of dilutions and incubation times for each new localization 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 and contrasted according to standard procedures.
— For double marking using secondary antibody gold conjugates, two primary antibodies produced in two different animal species are mixed and applied simultaneously (step 4).
After the washing step, a mixture of the corresponding gold conjugated reagents with two non-overlapping sizes is applied (step 6).
— For double marking using protein A or protein G gold conjugates each labeling is worked out separately.
An incubation with free protein-A or protein-G at a concentration of 20-100 µg/ml for 10-20 minutes is inserted after the first gold reagent incubation. Steps 4 through 6 are then repeated using a different size gold reagent.