AURION IMMUNOGOLD REAGENTS EDITION VI FREQUENTLY ASKED QUESTIONS


A second possibility is to use one-step incubations with a mix of primary antibodies, each labeled directly with a different gold particle size. We offer a custom labeling service. Details can be found in the section on custom labeling.


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 a flattened loop to transfer grids from droplet to droplet during immuno incubations. We do sell a perfect-loop (70944) for this application.


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.


• On occasion in the past, using OSO4 fixation after silver enhancement used to lead to the removal of part of the deposited silver. However, with the introduction of SE-EMplus


this is no longer the case, as the resulting


enhanced particles are no longer sensitive to oxidation. 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.


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. We have developed a chemically modified BSA especially for this purpose. Newsletter #1 gives in-depth information regarding it.


How can I do a controlled silver enhancement with pre- embedding?


With pre-embedding there are 2 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.


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 lg-types from different species which, when used as normal serum additive, would lead to an impressive amount of gold particles all over the specimen. We offer several Blocking Solutions tailored for specific secondary antibody or protein A/G incubations.


In which case should I use a Single Fab or F(ab’)2 conjugate instead 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 lg-molecule.


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