UranyLess EM Stain – Protocols of Use and FAQs

EMS22409

CLASSIC CONTRAST

This protocol is used for double staining with UranyLess / Lead citrate on ultrafine sections.

This protocol is adapted to biologic samples that have been fixed with glutaraldehyde, osmium, or ruthenium and embedded in an epoxy type resin (Epon, Araldite, Spurr) or acrylic type (LRWhite, HM20).

Staining protocol:

1. Place a drop of UranyLess on parafilm or any other hydrophobic slide.
2. Place the grid on the UranyLess drop for 1 to 2 minutes.
3. Blot the grid on a filter paper and then wash in room temperature distilled water.

Let it dry.

After drying, go to the lead citrate staining according to Reynolds method (1963).

1. Place the grid on the lead citrate drop according to the Reynolds method, for 1 minute.
2. Blot the grid on a filter paper before rinsing with room temperature distilled water.
3. Let it dry.

Technical Tip:

• UranyLess is not air or light sensitive, unlike Uranyl Acetate.
• After lead citrate, drain immediately in a freshly prepared room temperature distilled water bath or wash with 0.01N of NaOH solution. Cold water is charged with CO₂ and causes precipitate on the grid.
• If there is a precipitate in the solution, filter it prior to use.
• If solution was refrigerated, allow solution to return to room temperature prior to use.
• Do not keep lead citrate refrigerated.

NEGATIVE STAINING

Negative staining is a very useful technique in electron microscopy. It allows characterization of isolated particles of morphology as bacteria, virus, protein, nanoparticles, liposomes, exosomes, etc.

Staining Protocol #1:

1. On a piece of parafilm or any other hydrophobic carrier, place a drop of your solution (~ 10µl) and a UranyLess drop.
2. Using our fine tweezers, place your sample drop on a formvar-carbon coated grid. for about 1 minute.
3. Blot your grid using filter paper.
4. Place your grid on the UranyLess solution for 1 minute.
5. Blot, let it dry for 5 minutes and observe under the microscope.

Technical Tip:

• If the staining is too intense, wash with room temperature water for 1 minute.

Staining Protocol #2 (Debra M. Townley, Baylor College of Medicine):

1. One drop of suspension of 300 mesh formvar coated grids – 30 minutes
2. Wick away excess suspension with paper arrow (Watman No.1)
3. Place one drop of Uranyless on the grid before it is completely dry
4. Stain for 3 minutes
5. Wick away excess stain with paper arrow (Watman No.1)
6. Allow the grids(s) to dry completely before examination on the TEM
7. Times can be altered (suspension time, stain time) to suit the "look" you desire

FREQUENTLY ASKED QUESTION

What is UranyLess made from?

UranyLess is a solution ready for use, a mix of lanthanides (rare-earths).

How is UranyLess sold?

In an aqueous solution (water).

What is it's shelf life?

One year.

What are the storage conditions for UranyLess?

Store it at room temperature away from direct sunlight.

Does it need to be diluted?

No, it is sold ready for use.

What is its pH?

UranyLess pH is 6.8-7.

How to stain with UranyLess?

Simply drop UranyLess on your grid, and wait a minute. Dry, then contrast with lead citrate according to Reynolds method.

How do I avoid precipitates (dirt) on the grids?

Precipitates form when lead citrate is washed with cold water which is loaded with CO₂. Use water that has never been at low temperature and never store lead citrate at low temperature.

Is it the same protocol for every kind of tissue (animal, plant, marine)?

Yes it is - a double stain of UranyLess plus Lead citrate.

Does it adjust to every kind of resin?

Yes, it operates with every kind of resin (Epon, Araldite, Spurr).

Can it be used on negative staining?

Yes, it can be used on negative staining.

Can it be used for bloc contrast?

Some tests are in progress.

Is it adapted to a cryo use?

No, because it is prepared in water. However, we are currently developing many formulations of UranyLess, including ethyl UranyLess and acetone UranyLess, the latter being the best-adapted to cryogenic use.

Is it efficient on marine material?

Yes.

How is UranyLess packaged?

We sell UranyLess in an airless 30ml bottle and also in a brown 200ml bottle.

How does an airless bottle operate?

Its use is very simple; simply push on the head of the bottle to get a drop. When you release, the bottle back pump actuator lifts up. It prevents any air inlet in the bottle.

What is the advantage of an airless bottle?

It is a bottle in which air never enters. Some products, such as lead citrate, are atmospheric CO₂ sensitive. Thanks to this system, those products have a longer shelf life. It also allows the product to be deposited drop by drop, quickly, cleanly and in any position.

Can it be used with automated staining equipment?

Yes, the 200ml bottle is available for use with automated staining equipment. When using UranyLess for automated staining, do not wash longer than 10 minutes or you run the risk of losing all contrast.