Protocol of Use
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:
- Place a drop of Urany-Less on parafilm or any other hydrophobic surface.
- Place the grid on the drop of Urany-Less (1 to 2 minutes).
- Blot the grid on filter paper before carefully rinsing with distilled water.
- Let dry.
- After drying, enhance the contrast with lead citrate according to Reynolds (1963):
- Place the grid on the drop of lead citrate using Reynolds’ method in a sodium-saturated atmosphere (1 minute).
- Blot the grid on filter paper before carefully rinsing with distilled water.
- Let dry.
Warnings:
- UranyLess is neither air nor light sensitive, unlike Uranyl Acetate.
- After the lead citrate bath, rinse in freshly prepared distilled water, as water absorbs atmospheric CO2. Alternatively, wash with water containing 0.01 N NaOH.
- Never wash in water containing sodium hydroxide during the first contrast with UranyLess.
- If a precipitate is present in the solution, be sure to centrifuge or filter it before use.
- Allow the Urany-Less solution to return to room temperature for a few minutes if stored in the refrigerator to ensure consistent staining times.
- Do not store lead citrate in a cold environment as it absorbs CO2 and becomes gaseous.
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