Light Microscopy Immuno Incubation Protocol

General Remarks LM

Recommended Incubation Solution:

This buffer system is recommended both for secondary antibody reagents as well as for protein A, protein G and streptavidin reagents. Buffers are preferentially prepared fresh and can be used for periods up to two weeks.

Reagents

AURION Ultra Small Immunogold Reagents are the reagents of choice for the localization of extracellular and intracellular antigens in light microscopy.

The degree of penetration of immuno reagents into the cell interior depends on size of the reagents, specimen characteristics and (aldehyde)fixation. For the localization of intracellular antigens AURION Ultra Small Immunogold Reagents occasionally require a permeabilization with e.g. Triton™-X-100® in the first washing step immediately following fixation. In this way difficultly accessible intracellular antigens can be localized.

Marking set-up

Living cells are preferably incubated at 0-4°C or in the presence of 0.05-0.2% NaN₃ in order to prevent internalization of reagents.

Monolayers on coverslips are easily incubated using 6-well culture plates (Falcon, Nunc etc.) The glass coverslips are placed in the well and covered with incubation media (approximately 100 µl). During washing the coverslips are covered with 2 ml of washing medium and left on a rocking table.

Cell suspensions are gently pelleted after each incubation step. The pellets are resuspended in the medium used in the next step and the centrifuge tube containing the suspension is left on a rocking table.

On occasion efficient background suppression is obtained by using 1-10% heat inactivated Human AB-serum as additive to the incubation buffer.

Permeabilization

Whenever a permeabilization step should be necessary the following procedure may be employed: Triton&Trade;-X-100® is added in a final concentration of 0.1 - 0.5% to the washing buffer used immediately following fixation. This permeabilization step should last between 10 and 20 minutes while shaking gently.

NOTE: The use of more or less apolar fixatives (e.g. based on methanol, acetone, ethanol) already infers a limited degree of permeabilization to specimens as part of the lipid is removed.

Procedures for marking

Procedures can e.g. be found in the CRC Press-edition “Immunogold Labeling in Cell Biology”, A.J. Verkley & 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.

Immuno Incubation Procedure

Using Ultra Small Immunogold Reagents (subnanometer gold particles)

A rocking table is recommended for facilitated penetration and reagent exchange.

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 access 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 localization 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-LM as described below.

Silver Enhancement Procedure

Using AURION R-Gent SE-LM (Silver Enhancement for Light Microscopy)

1. Once the developer and enhancer reagents have reached temperature equilibrium, equal parts are mixed immediately before applying the enhancement mixture to the specimens. The specimens should be fully covered by the mixture.

2. Enhancement is done at room temperature (preferably 20°C). Gentle shaking is optional.

3. Typical enhancement times are between 15 and 25 minutes. Auto-nucleation is visible only after 30-40 minutes.

The on-going process may be monitored using an inverted light microscope with dimmed light conditions. In this way heat transfer to the enhancement mixture is kept low.

4. When enhancement is complete (judged by the presence of the brown to black signal developed) the specimens are washed extensively with distilled water (at least 3x5 minutes). A postfixation with photographic fixer is not required.

5. After washing light microscopical specimens may be counterstained according to standard procedures.

6. Short term storage (up to 1 year) can be achieved by mounting in water compatible Glycergel™ (DAKO).

For long term storage it is necessary to dehydrate the silver enhanced specimens and to mount in water-incompatible media such as Pertex (HistoLab, Sweden).