AURION IMMUNOGOLD REAGENTS EDITION IV FREQUENTLY ASKED QUESTIONS


We have collected answers to frequently asked questions from immunogold users. They are listed below. If your question is not listed on this page or you have further queries, please contact us directly by phone or e-mail. We remain at your disposal.


What kind of particle size should I use?


Always use the smallest particle size to fit your application. Conjugates based on smaller particles are more efficient than larger particle based conjugates. If visualization is difficult with smaller particles these can be enlarged with silver enhancement. Very sensitive specimens for SEM observation are best served with a larger particle size conjugate. This prevents ultra structural enhancement reagents.


Is it true that gold conjugates are more background prone than other conjugates?


No! This fairy tale comes from the fact that gold conjugates are based on particles and that visualization is also based on separate particles. Contrary to enzyme and fluorescent markers, gold conjugates are more like a digital system, either they are there and then you will see them, or they are not present. Enzyme and fluorescent markers are sooner to be considered as “analogue” markers, their visibility in detection increases with their local concentration or with the time the enzyme marker can produce a visible reaction product. An unbiased look at controls in fluorescence shows always a low level of light that is inherent to the presence of double bonds in biological compounds and on top of this comes the fluorescence from the labeled antibodies. Likewise will an unbiased look at control specimens incubated only with alkaline phosphatase or peroxidase labeled antibodies usually show a faint overall staining of the specimen. Such faint levels are easily accepted or even filtered out. You cannot do this with gold conjugates since they are based on particles.


Should I use a secondary gold conjugate or Protein A (or G)?


That depends on what your goal is. Using secondary conjugates results in a higher labeling density. Therefore it is often said that secondary conjugates are more sensitive than Protein A conjugates. This is partly true. Protein A (or G) recognizes only one site on a primary antibody molecule. Binding will occur only when this site is available and not obscured by its environment. Secondary conjugates recognize more sites on primaries and therefore the chance that a primary antibody will be detected is greater. Essentially this is the increase in sensitivity.


If all primary antibodies would be available to the sane extent for binding to either Protein A or a secondary antibody conjugate, then the use of the latter would only result in more particles. This helps in localizing antigens at low magnifications, in other words this is an increase in detectability.


Is there a training program for immunogold (silver) staining where I can bring my own specimens?


EMS and Aurion organizes wet-workshops in Europe and the USA 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 two days and give an in-depth view in immunogold (silver) staining. Detailed information on the setup of our workshops can be found in this publication.


There are a few newsgroups which may be of interest: bionet.cellbiol, bionet.cellbiol.cytonet, bionet.molbio.methds- reagnts and sci.bio.immunocytochem. There is a microscopy listserver 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 words "subscribe microscopy".


Is it possible to do pre-embedding labeling of intracellular antigens?


Yes. Single cells are most suited. Plant material with a thick impenetrable wall is not. The Ultra-Small gold conjugates are the conjugates of choice. In many cases a permeabilization step with NaBH4 suffices to open up the specimens and allow penetration of reagents. Low concentrations of mild detergents like saponin help. One thing should be emphasized: reaction times have to be prolonged since full penetration of the reagents to the internal antigens has to be achieved. To remove unreacted reagents after incubation wash procedures have to be adapted likewise! The Aurion Newsletter #5 deals with this topic.


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. 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.


How can I verify that the silver enhancement reagents are still fine?


Again, there is a simple procedure to check this. It is described in great detail in our Newsletter #4. In short: you need a nitro-cellulose strip, apply dots from a dilution series of your gold conjugate and incubate the strip with the silver enhancement reagents. The dots should become brown- black. During this period of time the mix of reagents should remain glass clear without any visible presence of silver caused by auto nucleation.


The activity of the Silver Enhancement reagent SE-EM for Electron microscopy can be tested by adding 10µl of the enhancement mix. The solution s should turn yellow in 30-45 minutes.


Is it advisable to use outdated conjugates?


As long as their reactivity is OK and there are not too many clusters formed this is no problem. Gold conjugates are very stable. There may be some release of protein from the particle surface with time, but generally this does not result in reduced reactivity. The reactivity of the conjugate is easily checked with a dot-spot test as described in Newsletter #4. Cluster formation may increase with time, depending on the type of conjugated protein and the particle size. The larger the particles the more clusters. These can be removed by centrifugation of the diluted conjugate before use.


Is it possible to double label using two antibodies from the same animal source?


Yes, there are ways to do this. One is by using Protein G or Protein A conjugates with different particle sizes. The procedure would be: first incubate with primary antibody l, detect this with Protein A (or G) with the smaller particle size. Then incorporate an incubation with excess free Protein A or G (50-100 µg/ml). This will block practically all binding sites for Protein A or G. Next, incubate for the second antigen with primary antibody ll and detect this with the larger sized Protein A or G gold conjugate.


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