Immunogold - frequently asked questions
What kind of particle size should I use?
Always use the smallest particle size to fit your application. Conjugates basedon smaller particles are more efficient than larger particle based conjugates. If visualization is difficult with smaller particles these can be enlarged with silver enhancement, which is a must for the conjugates from the Ultra Small series. The new silver enhancement system AURION SE-EM provides for homogeneous and high efficiency enhancement.
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 mentally 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 therefor the chance that a primary antibody will be detected is greater. Essentially this is the increase in sensitivity.
Is there a training program for immunogold (silver) staining where I can bring my own specimens?
EMS and Aurion organize wet-workshops 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 or three days and give an in-depth view in immunogold (silver) staining. The number of participants is limited to warrant optimum teaching. You may contact us directly for more info. Detailed information on the setup of our workshops can be found in this website.
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. Please see elsewhere on this site.
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 "Newsletter" 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.
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 (please refer to the "Newsletter" section of this web site). 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 diluted ultra small reagent to 100µl of the enhancement mix. The solution 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 noticeably 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 I, 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 II and detect this with the larger sized Protein A or G gold conjugate. A second possibility is to use one-step incubations with a mix of primary antibodies, each labeled directly with a different gold particle size. Custom labeling services are available.
What kind of grids should I use for silver enhancement?
Nickel is the material of choice. Gold grids are out of the question as they will be neatly enhanced as well. The same with copper. Nickel grids are preferred to copper ones for immuno incubations anyway, since nickel is more inert and less poisonous to immuno or enzyme reactions. Nickel grids can be annoying because of their magnetic properties. This is easily overcome by using either non-magnetic tweezers or by using the Electron Microscopy Sciences "perfect loop" to transfer grids from droplet to droplet during immuno incubations.
What about silver enhancement and OsO4?
OsO4 fixation can be used before incubation, after incubation or after silver enhancement.
Because of its destructive effect on antigens OsO4 fixation is not often used when immuno incubations are intended. However, in general silver enhancing immuno incubated OsO4 fixed specimens causes no difficulties.
An Osmium fixation step can be introduced after incubation to improve contrast in specimens. As stated before, applying silver enhancement generally causes no difficulties.
Using OsO4 fixation after enhancement is also possible but since OsO4 is a strong oxidant it is capable of oxidizing metallic silver, especially when present as particles. This results in removal part of silver. A simple remedy is to combine slightly over-enhanced specimens with a limited OsO4 fixation, for example 1% OsO4 for 15 minutes.
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 "Newsletter" 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:
Residual fixative activity, which is eliminated by using a NaBH4 or Glycine block step prior to the protein block step, 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,
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. Newsletters are available online here.
How can I do a controlled silver enhancement with pre-embedding?
With pre-embedding there are two possibilities: either the enhancement is done before embedding or on the sections after embedding. We prefer to do the enhancement on sections (on nickel grids) since this gives more control over the degree of enhancement. Using longer enhancement times allows to observe larger (even ultra thin) sections in the light microscope. This facilitates searching for the area in the specimens where a reaction has occurred and allows easy targeting and trimming down to the area of interest for EM sectioning. Shorter enhancement is then used on sections for EM. Using enhancement before embedding has the disadvantage that once enhancement proves to be too long (resulting in too large particles) this can not be reversed.
In which case should I use a Single Fab or F(ab)2 conjugate in stead of the complete immunoglobulin conjugate?
The size of a conjugate is co-responsible for its efficiency. The overall size is determined by the particle size and by the size of the proteins adsorbed onto the particle surface. That is why we introduced ultra small particles in the first place. Whenever a specimen is relatively dense or intensely cross-linked immuno reagents will be more hindered in their action. If you are already using an ultra small conjugate further improvement may result from using a single Fab or F(ab)2 fragment of the specific secondary antibody instead of the intact Ig-molecule.
When should I use normal serum in the incubations?
It is a good idea to use normal serum as an additive to the blocking and incubation buffer when using secondary antibody conjugates. The normal serum should be the same species as the secondary antibody conjugate. Its action is similar to the action of BSA. Please be careful when using normal sera to suppress background with Protein A or Protein G conjugates. These conjugates detect several Ig-types from different species which, when used as normal serum additive, would lead to an impressive amount of gold particles all over the specimen. Aurion offers several Blocking Solutions tailored for specific secondary antibody or protein A/G incubations.
What about sensitivity, signal-to-noise ratios and detectability?
Sensitivity can be considered at different levels in the total of preparation and incubations. Ideally during preparation one would like to preserve all antigens present. In many cases this is not possible. But at least a representative fraction should be preserved and be available for immuno labeling. It all depends on the preparation procedure (fixation, embedding, temperature, etc.), which leaves you with a specimen or section with a given number of available and recognizable antigens. The ensuing detection protocol has 100% sensitivity if all the remaining antigens are detected, i.e. are represented by at least one gold particle or marker molecule. Again, due to masking and steric hindrance by the specimen composition this will only in exceptional cases be fully attained. The immuno labeling sensitivity thus expresses the degree to which available antigens can be detected by the employed combination of primary antibody and secondary conjugate.
The quality of the primary antibody is the next important item. Theoretically the Kd-value of an antibody/antigen reaction is a measure for the dilution at which the incubations should be performed and for the stability of the ensuing bond. Sensitivity will go up with more concentrated antibody solutions up to a maximum level. However, when the primary antibody shows cross-reactivity there is not necessarily an improved signal-to-noise ratio. The reliability of the detection by the primary antibody improves in such cases with higher dilutions, probably leading to a smaller amount of antigens detected, but to an improved signal-to-noise ratio. Thus, sensitivity at the level of the primary antibody has to be balanced against the signal-to-noise ratio.
The last step is the quality of the secondary reagent. In fact you will be looking at a number of gold particles which represents a number of secondary antibodies which have detected a number of primary antibodies. For the interaction between the secondary reagent and the primary antibody the same rules apply as indicated for the antigen/primary antibody reaction.
Detectability reflects the degree to which the final result of all the reactions involved can actually be seen. This is depending on the right match between particle size and magnification. Ultra small particle-based conjugates for instance are among the most efficient detection systems, but you will only detect them after silver enhancement (in most applications).
What is epi-polarization microscopy?
Epi-polarization is a technique used for the very sensitive light microscopical observation of metal particles. Where bright field microscopy depends on contrast levels in discriminating signals, epi-polarization works differently: provided particles are large enough individual particles will be observed. So in fact you are evaluating your labeling results on the same basis as with an electron microscope by looking at individual particles. This makes this technique so valuable as it builds a bridge between the light level and the electron microscopical observation.
What do you need to do this: a high-quality light microscope equipped with an epi-illumination source, preferably a high pressure Hg-lamp (although a halogen source may also do). Many laboratories have an epi-fluorescence microscope at their disposition with a 40X (or higher) oil objective. Such microscope equipment forms the correct basis. You only need to implement an epi-polarization filter (the so-called epi-block or IGSS filter) in the filter housing. The epi-block contains two polarizers, differing 90 degrees in orientation with respect to each other.
How does it work (in short): High intensity light passes the first polarizer in the epi-block and becomes polarized. The polarized incident light passes the objective lens and interacts with the specimen. The biological material hardly gives any reflection, and the reflected light is unmodified. The metal particles mirror the polarized light, thereby randomizing the polarization angle. Reflected light passes up through the objective lens. On its way to the eye pieces or the photo camera the light passes the second polarizer in the epi-block. While doing so, light with the original polarization angle (the way it was polarized in the first place before ever hitting the specimen) is extinguished, whereas light that has become randomly polarized (and which comes from the silver metal particles) passes the epi-block. As a result you will see individual bright stars (the gold/silver particles) against a dark background.
Epi-polarization observation can be combined in real time with bright field imaging, providing for a very sensitive detection of even extremely low amounts of antigen while still having the advantage of full morphological details in the specimen.
My specimens for pre-embedding have a lot more antigens than a corresponding ultrathin section. Should I use more concentrated reagents?
No, the higher amount of antigens should be balanced by a larger amount of reagent volume at an appropriate dilution (the same as used on sections with low amounts of antigen), and not by more concentrated reagents. The reason is that with increased concentrations more cross-reactions may occur and signal-to-noise ratios will decrease.
Incubating specimens for pre-embedding in larger volume quantities is best performed on a rocking table for a prolonged time to warrant penetration to antigenic sites in the specimen.
What is the advantage of gold nanoparticleconjugation via adsorption?
This is an easy and straight forward method for the conjugation of highmolecular weight macromolecules. The flocculation test that tests the effect of a high salt concentration on the stability of the complex makesit easy to check if adsorption to the gold nanoparticle surface issuccessful or not.
Can you give me more information on goldnanoparticle conjugation strategy?
First check if conjugation via adsorption is feasible. If yes, check bioactivity e.g., via a dot spot test. Biomolecules that are too small to beconjugated via the classic adsorption method can be covalently linked to functionalized gold nanoparticles as an alternative. A prerequisite forsuccessful conjugation to Carboxyl-functionalized gold nanoparticles isthe presence of primary amine(s) in the target molecule. As a rule ofthumb conjugation via classic adsorption to conventional goldnanoparticles is possible with macromolecules having a MW > 40 kDa.
What is the conjugation principle behind covalentconjugation using carboxyl-functionalized goldnanoparticles?
The conjugation relies on the well known and proven EDC/sulfo-NHSchemistry. EDC/sulfo-NHS activation at pH 5 results in an amine-reactive ester, immediately followed by binding to free amine on thetarget molecule