All version of EM Stain Uranyless

UranylessUranyless Cryo AFS Uranyless Alcohol
SolventH2O water DistiledAcetoneEthanol 30%
Use Contrast agent for ultrathin sections or negative stainingBlock staining for AFSStaining of dense and lipid-rich tissues
Cryofixation 
Technique Manual StainingAutomatic staining for EM stain Leica or RMC grids stainerManual StainingManual StainingAutomatic staining for EM stain Leica or RMC grids stainer
Volume 30ml 200ml 100ml 30ml 200ml 
Reference 11000 11000-200 11000C-100 11000E-30 11000E-200 

Uranyless

EM Stain Uranyless aqueous is a contrasting solution that substitutes for uranyl acetate. As recommended by Reynolds (1963), it is highly advisable to reinforce the initial contrast (in our case, UranyLess) with lead citrate in a NaOH-saturated atmosphere to prevent contamination from atmospheric CO2.

To facilitate the use of lead citrate during contrasting, it is packaged in AirLess bottles (without air and thus without CO2). You can store this 30 ml bottle for a long time without the risk of CO2 contamination, a common problem for microscopists.

The 30 ml AirLess bottle packaging allows you to contrast over 1500 grids.

This packaging is doubly effective: it provides long shelf life and generates minimal waste.

For using UranyLess aqueous in automated contrast devices like Leica’s EM Stain, UranyLess is available in a 200 ml bottle. It is worth noting that UranyLess aqueous is packaged in an AirLess bottle to facilitate drop dispensing only, as it is not affected by air or light.

Protocols of Use for Classic Contrast

Classic Protocol: Protocol for Using Double Contrast on Ultrafine Sections with UranyLess / Lead Citrate This protocol is suitable for biological samples previously fixed using conventional methods such as glutaraldehyde, osmium, ruthenium, and embedded in epoxy (Epon, Araldite, Spurr) or acrylic (LRWhite, HM20, etc.) resins.

Contrast Protocol:

  1. Place a drop of Urany-Less on parafilm or any other hydrophobic surface.
  2. Place the grid on the drop of Urany-Less (1 to 2 minutes).
  3. Blot the grid on filter paper before carefully rinsing with distilled water.
  4. Let dry.
  5.  After drying, enhance the contrast with lead citrate according to Reynolds (1963):
  6. Place the grid on the drop of lead citrate using Reynolds’ method in a sodium-saturated atmosphere (1 minute). 
  7. Blot the grid on filter paper before carefully rinsing with distilled water. 
  8. Let dry.

EM Stain Uranyless protol of classic contrast

Negative Staining

Negative staining is a very useful technique in electron microscopy that allows for the characterization of the morphology of isolated particles such as bacteria, viruses, proteins, nanoparticles, liposomes, exosomes, etc.

Staining Protocol:

  1. On a piece of Parafilm or any other hydrophobic surface, place a drop of approximately 10 µl of your solution and a drop of UranyLess.
  2. Using fine tweezers, place a grid coated with a formvar-carbon film on the drop of your sample. Leave in contact for about 1 minute.
  3. Drain the grid using filter paper.
  4. Contrast the grid by placing it in contact with the drop of UranyLess for 1 minute.
  5. Drain, dry for 5 minutes, then observe under the microscope.
 

Fine-Tuning the Technique:

  • Sometimes you may have very little sample on your grid; in this case, ionize your grid.
  • If you have too much sample, wash it on a drop of water before contrasting.
  • If the staining is too intense, wash for 1 minute in water after contrasting with UranyLess.

Uranyless Cryo

Uranyless anhydrous in acetone is primarily used for cryogenic methods, such as AFS cryo-substitution. This solution is ideal for applications where it needs to be mixed with fixative solutions like PFA, Glutaraldehyde, or OsO4, and is particularly suitable for cryo embedding in resin.

Protocol:

  1. Preparation: Mix anhydrous Uranyless with 100% acetone.
  2. Wash: Wash with 100% acetone twice for 30 minutes each.
  3. Contrast Application: Use anhydrous Uranyless for cryo in acetone.

Additional Notes:
Uranyless in acetone is a solution of a lanthanide mixture and is specifically formulated for cryofixation AFS applications. When used in combination with OsO4, it is recommended to follow a specific protocol to avoid undesirable interactions that could reduce the effectiveness of Uranyless.

 

Uranyless Alcohol

Uranyless in ethanol is used for contrasting very dense and lipid-rich tissues that are difficult to contrast with other agents. The 30% ethanol solution allows the contrast to penetrate well, especially in tissues rich in tightly packed collagen, such as the cornea. This concentration is a sufficient compromise to penetrate deeper without dissolving lipid structures.

Protocol:

  1. Contrast: Apply Uranyless/ethanol for 2 to 3 minutes.
  2. Wash: Rinse with 30% alcohol for 1 minute and blot.
  3. Additional Contrast: Apply lead citrate #11 300 (Reynolds) for 1 to 2 minutes.

Additional Notes:
Uranyless is prepared in 30% ethanol with water, which helps the contrast penetrate better, particularly in tissues rich in tightly packed collagen.

EM Stain Uranyless alcohol protocol

FAQ

UranyLess aqueous is a ready-to-use solution based on a mixture of lanthanides (rare earths).

Currently, UranyLess is only marketed in aqueous form (water).

Its shelf life is unlimited. With a 30ml bottle, you will have about one year of use.

It is advised not to refrigerate this product but rather to keep it at room temperature. It is not sensitive to air or light.

If your tissues rich in lipids such as nervous, adipose tissues, some plant tissues show weak contrast, reduce water washes. Unlike uranyl acetate, which is contrasting and fixing (making it a toxic agent in addition to radioactive), UranyLess is not a fixative and does not bind and is more easily washed away with water during washing (so reduce washing times after contrasting with UranyLess.

Yes, it works with all resins (Epon, Araldite, Spurr…).

Yes , we have now  tested new uranyless solved in 100% acetone anhydrous (glass distilled) adapted Cryo special for on bloc staining when you used AFS (see protocol)

We market UranyLess in 30ml in an airless bottle as well as in 200ml in a brown bottle.

Its use is very simple, just press the head to release a drop. When you release, the push pump at the bottom of the bottle will rise. This prevents any air from entering the bottle.

UranyLess is sold ready-to-use. There is no need to dilute it.

The pH of UranyLess is 6.8, unlike uranyl acetate, which is at 4.

Simply place your grid on a drop of UranyLess and wait for a minute. Dry, then contrast with lead citrate according to Reynolds’ method. You can access the protocol in the Technical Documents section or by clicking on the following link: The Classic Contrast.

Yes, the double contrast UranyLess – Lead Citrate is used.

Tests are ongoing.

Yes. The protocol can be seen on the Negative Staining page.

Yes, UranyLess provides good contrasts.

UranyLess waste should be disposed of in heavy metals (Rare Earths). Please consult the Safety Data Sheet for more information.

Firstly, it is a bottle into which air never enters. Some products, like Lead Citrate, are sensitive to atmospheric CO2. Thanks to this system, these products have a longer shelf life. Moreover, it allows the product to be dispensed drop by drop, quickly, cleanly, and in any position. For more information, please consult our commercial offer.

 
 
 
 
 
 

Originally our classic uranyless which is the most used is based on distilled water. At the request of several users, we have developed a uranyless for cryo AFS (staining on bloc) based on anhydrous acetone and Uranyless based on ethyl alcohol / water 30% to stain very dense fibrous tissues, with this uranyless Alcohol the contrasting agent penetrates deeply and allows a better result.

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