Streptavidin - Gold Conjugates

Contents
Introduction Methods References

Introduction
The usefulness of streptavidin-gold conjugate is due to the high affinity of streptavidin for biotin (dissociation constant of 10-15M). Streptavidin has four binding sites for biotin but fewer than four molecules will actually bind. Biotinylation of a primary antibody or nucleic acid probe is a mild process where biotin is covalently linked to the protein or probe. The streptavidin conjugate then binds to the biotin during incubation and provides a visible gold label that may be directly viewed in the EM or observed in the LM (or EM) after silver enhancement. Any gold particle size may be employed for EM studies. Particles of 1nm will be most easily viewed if silver enhanced for 3-5 min (on the EM grid) as below. For LM studies silver enhancing (5-10min) is necessary whether using 1nm or 5nm gold conjugates. In both cases the sections may be counterstained after silver enhancing. The sequence of reagent application depends on whether a single primary antibody or the secondary antibody is biotinylated. Whichever method is chosen it may be followed by the streptavidin gold conjugate. The sections are then washed and viewed, or silver enhanced and viewed. Excellent results can be found on fixed, paraffin embedded specimens as well as on cryo sections and cells in suspension. Biotin is a vitamin and coenzyme and may be found in a wide variety of tissues. Some tissues such as liver, kidney and brain contain endogenous biotin and must be blocked to avoid non specific staining. This is most pronounced when using frozen sections but can be overcome by adding excess streptavidin to block the endogenous biotin. No details are given here on specimen fixation or embedding since these depend on each specific application. Please refer to our Technical Services Department and to the bibliography listed at the end.

Methods Double antibody method (LM or EM), for sections.

Wax embedded LM sections must be dewaxed and brought to water. Cryostat sections must be thoroughly air dried, fixed (e.g. acetone) and air dried again before labelling. EM sections are mounted on nickel or gold grids. For LM incubations reagents (50l) are applied to the slides, covering the sections. For EM incubations sections on grids are floated on droplets of reagents (50l). Steps 3-6 are used to block endogenous biotin if necessary. Otherwise proceed directly from step 2 to step 7. The same buffer may be used throughout for washing and dilutions. PBS or TBS containing 1% BSA at pH78.5 is normally used. See Choice of Buffers for details on suppression of background and signal enhancement by correct choice of buffer and additions. It is important that the sections are not allowed to dry during step any of the incubations. 1. Gently rinse the section (slide or EM grid) thoroughly with buffer. 2. Remove excess liquid from around the section. Do not dry. Blocking if necessary 3. Apply excess streptavidin (0.1%) to the section for 20 minutes to block endogenous biotin. 4. Repeat steps 1 and 2. 5. Apply excess biotin (0.01%) to cover the remaining binding sites of streptavidin for 20 minutes. 6. Repeat steps 1 and 2. 7. Apply the primary antibody (unlabelled) diluted as appropriate for 30-60 min. 8. Repeat steps 1 and 2. 9. Apply the biotinylated antibody diluted as appropriate for 30-60 min. 10. Repeat steps 1 and 2. 11. Apply streptavidin: gold conjugate diluted as appropriate for 60 min. 12. Repeat steps 1 and 2 13. Apply 1% glutaraldehyde in PBS for 5 minutes. This fixes the antibody / streptavidin conjugate to the section. 14. Repeat steps 1 and 2. 15. Wash thoroughly in distilled water to remove all traces of buffer. 16. EM sections may be counterstained and examined immediately. Otherwise the gold particles may be silver enhanced (especially 1nm gold) as for LM

sections below. 17. Apply the BBlnternational Silver Enhancing solutions for 3-5 min (EM sections) or 5-10 min (LM sections). Observe the enhancement in the microscope (LM sections). 18. Wash thoroughly in distilled water to terminate the enhancement. 19. If necessary continue the enhancement in fresh solutions. 20. Counterstain and examine in the microscope (EM and LM). Dilutions of antibodies must be determined experimentally. Typical dilutions are as follows: Primary (polyclonal) 1/100 - 1/1000 Primary (monoclonal) 1/10-1/200 Secondary (biotinylated polyclonal) 1/100 - 1/500 (according to manufacturers recommendations). Streptavidin: gold conjugate 1/50 - 1/400.

Single antibody method (LM or EM) for sections

The method is the same as above except that the primary antibody is biotinylated and no secondary antibody is used. Omit steps 9 and 10.

Blood Cells
Cells should be thoroughly air dried (1-2 hours) onto the glass slide and fixed by normal methods. The slides are then treated as above. If necessary the membranes may be further permeabilised for detection of intracellular antigens.

Cytology smears
Cells are usually fixed on the slide while still wet with 95% ethanol and thoroughly air dried. This preserves the chromatin structure for evaluation of nuclear changes. The slides are then treated as above.

Cells in suspension (EM)

Cells in suspension may be labelled with biotinylated antibodies followed by streptavidin: gold conjugates and then embedded and sectioned for EM viewing as follows: 1. Gently fix the cells as appropriate in the suspension buffer. Typically use 4% paraformaldehyde and 0.1% glutaraldehyde for 20 min in PBS. 2. Wash in buffer by gentle centrifugation for 10 min. Resuspend and repeat the wash twice. Resuspend in buffer.

3. Incubate in primary antibody diluted as appropriate for 30 min. 4. Repeat step 2. 5. Incubate in biotinylated secondary antibody diluted as appropriate for 30 min. 6. Repeat step 2. 7. Incubate with streptavidin: gold conjugate for 30-60 min. 8. Repeat step 2. 9. Fix with 0.1% glutaraldehyde in PBS for 10 min. This fixes the antibodies / streptavidin: gold conjugate to the cells. 10. Repeat step 2. 11. Centrifuge the cells to a pellet. 12. Embed the pellet and process with normal procedures to sections for EM observation. If the primary antibody is biotinylated then steps 5 and 6 are omitted. Dilutions of antibodies and streptavidin: gold conjugate must be determined experimentally but typical dilutions are as above. The above protocols are intended as a guide only. No detailed information is given here regarding specimen fixation and embedding since these vary for each application. Please refer to the bibliography for more details on specimen preparation.

References
1. Bullock GR and Petrusz P (eds.) Techniques in Immunocytochemistry. Vols. 1-4. Academic Press, London, 1982,83, 85,89. 2. Polak J and Van Noorden SI (eds.) Immunocytochemistry: Modem Methods and Applications. Butterworth Heinemann Oxford, 1986. 3. Hayat M (ed). Colloidal Gold. Principles, Methods and Applications. Vols. 1, 2, 3. Academic Press, California, 1989, 91. 4. Polak J and Varndell I (eds.) Immunolabelling for Electron Microscopy. Elsevier Science, Amsterdam, 1984. 5. Baker JR. Principles of Biological Technique. Methuen, London, 1970. 6. Lillie RD. Histopathologic Technique and Practical Histochemistry (3rd edition). McGraw Hill, New York, 1965. Biotinylation kits and probes are available fromVector Labs. In Situ Hybridisation 1 - Detection of Digoxigenin and Biotin Labelled DNA Probes Using an Immunogold Silver Staining Technique

In Situ Hybridisation 2 - Detection of mRNA with Oligonucleotide Probes using an Immunogold Silver Stain Technique In Situ Hybridisation 3 - Some Practical Hints into the Technique of In Situ HybridisationIn Situ Hybridisation Publications