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 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 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 distilled water bath or wash with 0.01N of NaOH solution.
• 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 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. 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.

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 distilled water.
3. Let it dry.

Technical Tip:

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