HIST - UranyLess Protocols of Use

Classic Contrast 

This protocol is used for double staining with UranyLess/Lead citrate on ultrathin 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). 

Staining Protocol: 

1.  Place a drop of UranyLess on parafilm or any other hydrophobic slide. 
2.  Using fine tweezers, 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. 
4.  Let it dry. 
5.  Place the grid on the lead citrate drop according to the Reynolds method, for 1 minute. 
6.  Blot the grid on a filter paper and rinse with distilled water. 
7. Let it dry. 
   
   

Negative staining 

This protocol is often used in diagnostic microscopy for contrasting a thin specimen with an optically opaque fluid. In this technique, the background is stained, leaving the actual specimen untouched, and thus visible. 

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.  Use fine tweezers to 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 (1 minute). 
5.  Blot, let it dry for 5 minutes and observe under the microscope.
   
   If the staining is too intense, wash with room temperature water for 1 minute. Suspension time and stain time can be altered to achieve desired results.

NOTE: UranyLess cannot be adapted to cryo use because it is prepared in water.