Post-embedding Immuno Incubation Protocol

General remarks Post-embedding

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.

Labeling ultrathin cryosections

After transfer of sections to a (nickel) grid first the covering sucrose layer has to be removed. If immuno incubations are performed immediately after sectioning the grids are simply rinsed on a drop of the incubation buffer. Mostly, however,  grids are collected on a solidified 2% gelatine layer in buffer (corresponding to the incubation buffer) in a small Petri dish on ice. The sucrose layer which is facing the solidified gelatine is in this way allowed to diffuse gently away and to be at least partly replaced by incubation buffer. This procedure is supposed to be less destructive to the ultrastructure since concentration shocks are avoided. In this way sufficient grids can be collected and stored at 4°C if necessary overnight.If the grids with the sections were stored on a gelatine layer the closed Petri dish is warmed to 37°C for 30 minutes in order to liquefy the gelatine. Incubation buffer (37°C) is added in a 1:1 ratio to the liquefied gelatine.

On-grid labeling for electron microscopy

The use of nickel grids is recommended, especially if silver enhancement procedures are intended.

For most applications grids are floated on top of drops of immune reagents displayed on a sheet of Parafilm™. They are washed on larger drops of buffer. Whenever larger series of grids or coated grids need to be processed, the use of microtiter plates is preferred during incubations to avoid the risk of cross-contamination (e.g. Falcon 3034, Falcon Plastics, Oxnard, CA 93030, USA).

Transfer of the grids from droplet to droplet or from well to well can be performed with fine forceps. Preferably, a metal loop of 3.2 mm initial diameter made of nickel-coated copper wire of 0.2 mm thickness is used. The loops are flattened between flat beaks of a vice. The use of such loops diminishes the risk of contamination and greatly facilitates transfer of grids.

Procedures for marking

Procedures can e.g. be found in the CRC Press-edition “Immunogold Labeling in Cell Biology”, A.J. erkley & J.L.M. Leunissen eds., (1989), Boca Raton, Florida. In addition the issues of the Academic Press edition “Colloidal Gold”, M.A. Hayat ed., (1989), San Diego, California are highly recommended.

Protocol Conventional Reagents

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.

Double labeling

-- 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.

Protocol Ultra Small Reagents

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 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. 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 3x10 minutes). A postfixation with photographic fixer is not required.

4. Grids are contrasted according to standard procedures.