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.
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. Electron Microscopy Sciences offers an excellent “perfect loop” for this purpose.
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.
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 “Sharing our Knowledge” 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 when testing Aurion R-Gent SE-LM, the Silver Enhancement reagent for Light Microscopy. During this period of time the mix of reagents should remain absolutely clear without any visible presence of silver caused by auto nucleation.
The activity of the Silver Enhancement reagent Aurion R-Gent 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. R-Gent SE-EM may also be tested using the dot spot test as described in Newsletter #4. Spots turn to yellow in the same time span spots turn to brown- black when testing Aurion R-Gent SE-LM. Due to the lower contrast obtained with Aurion R-Gent SE-EM clear visibility is limited to the spots that contain higher protein concentration.