How can I verify that my conjugates are still active?
There is a simple procedure to check this. It is described in great detail in Aurion’s Newsletter #4 about which some information can be found in the “Sharing our Knowledge” section of this web site. In short: you need a nitro-cellulose strip, apply dots from a dilution series of your primary antibody and incubate the strip with the gold reagent. The dots will stain red with the larger conjugates. When testing an Ultra Small conjugate silver enhancement has to be applied for visualization.
I get no positive results, now what?
When your incubated specimens look as clean as your controls, either (one or more of) the reagents are inactive, or the antigens are destroyed, masked or absent. The cause is easily found by performing tests working backwards through the incubation protocol using dot-spot tests as described in Newsletter #4 (please refer to the “Sharing our Knowledge” section of this web site).
First test the activity of the silver enhancement reagents (if they were used at all) on the gold conjugate that was used. If silver enhancement is fine, the next step is to test the gold conjugate on the primary antibody used and so on. If it proves that the problem is not in the reagents, you will have to look into antigen preservation. Is a different fixation due? Or a different embedding medium? Using light microscopical evaluation of the results such questions are answered without tedious EM experimental work.
I am having background problems; is this due to the gold conjugate?
When specimens are blocked correctly and the right composition and condition of incubation buffer is used, background levels should not be interfering with specific signals. Some background will always exist: to some extent all compounds have a certain affinity for other compounds and depending on availability and concentration an interaction may occur. There is no absolute black and white in this respect.
When you leave out the primary antibody incubation and only use the gold step and your background has become much reduced, then your primary antibody causes background. Remedy: purify the primary antibody by either affinity chromatography (in case of an antiserum) and/or by cross-adsorption. If you have unacceptable levels of background without using a primary incubation, then the specimen has a tendency to bind to gold conjugates.
Background may have many causes which are centered around three different types of interactions:
1) Residual fixative activity, which is eliminated by using a NaBH4 or Glycine block step prior to the protein block step.
2) Stickiness to hydrophobic areas (embedding medium, lipid rich specimen compounds). This is reduced by using an adequate protein block step involving a partly hydrophobic protein like BSA or Casein.
3) Charge-based interactions causing negatively charged reagents such as antibodies and gold conjugates to adhere to oppositely charged areas in the specimen (notorious are the histone proteins, some collagen types and poly-L-lysine that is sometimes used to make sections stick to surfaces). This type of interaction can only be overcome by adding an excess of negatively charged indifferent molecules to the incubation media. Aurion has developed a chemically modified BSA termed BSA-c ™ especially for this purpose. Newsletter #1 gives in-depth information. Information on the Aurion Newsletters is available in the “Sharing our knowledge” section of the Aurion web site.
Is there a training program for immuno gold (silver) staining where I can bring my own specimens?
Aurion organizes workshops worldwide where you preferably work with your own specimens and primary antibodies. After all, that is where your interest lies. If required, we will expand our activities to additional venues. The workshops last for three days and give an in-depth view in immuno gold (silver) staining. The number of participants is limited to warrant optimum teaching. You may contact us directly for more info. Detailed information such as dates of forthcoming workshop and the setup of our workshops can be found in here.
Are there any fora which I can address with questions regarding labelling or microscopy?
Feel free to address our help desk by e-mail with questions regarding immuno labeling.
There are a few newsgroups which may be of interest such as: bionet.cellbiol, bionet.cellbiol.cytonet, bionet.molbio.methds-reagnts and sci.bio.immunocytochem. And last but not least: there is a microscopy list server to which you can subscribe and which offers a platform to ask questions regarding light and electron microscopy in all its facets. You can subscribe by sending an e-mail message to ListServer@MSA.Microscopy.Com. The message only has to contain the word “subscribe microscopy”.