4. Glass slide wrapped in parafilm or aluminum grid block.
5. Grid storage holder.
6. Glow-discharge system.
2.2 Stain Solution:
Preparation
and Storage
1. Uranyl Acetate (UA) or Uranyl Formate (UF) powder.
2. Balance and weighing paper.
3. Milli-Q or double-distilled water.
4. Tubes wrapped with aluminum foil (for storage).
5. Bunsen burner, tongs, and conical flask (to be used only for
boiling water).
6. 10 mL beaker for UF or appropriate volume beaker for UA.
7. Magnetic stir bars for beakers in #6.
8. 250 mL beaker wrapped with aluminum foil (or equivalent).
9. Stir plate.
10. Timer.
11. 5 M NaOH (filtered).
12. 0.22 μm filters and syringes.
2.3 Staining
Workflow
1. Parafilm.
2. Filter paper (Whatman #1).
3. Pipettes (capable of dispensing 50 μL and 2–4 μL).
4. Milli-Q water.
5. (optional) Vacuum system with a fine tip and filtered to block
the aspirated stain reaching the vacuum source.
3 Methods
Preparation of Uranyl Stains.
Important: UA and UF are light-sensitive, toxic, and radioactive. Adhere to appropriate health and safety procedures
(including use of a fume hood due to the stain powder) and
also separate stain equipment from other lab equipment.
3.1 Uranyl Acetate
(2%) (Concentration
Can be Adjusted)
Here, it is important to minimize light exposure of UA during the
following steps.
1. Weigh UA for a final 2% (w/v) and add to Milli-Q or doubledistilled water in a glass beaker with a stir bar.
2. Stir in the dark until fully dissolved.
3. Filter solution using a 0.22 μm filter and store in the dark. UA
solution can be stored at room temperature and used for
months.
Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples
119
5. Grid storage holder.
6. Glow-discharge system.
2.2 Stain Solution:
Preparation
and Storage
1. Uranyl Acetate (UA) or Uranyl Formate (UF) powder.
2. Balance and weighing paper.
3. Milli-Q or double-distilled water.
4. Tubes wrapped with aluminum foil (for storage).
5. Bunsen burner, tongs, and conical flask (to be used only for
boiling water).
6. 10 mL beaker for UF or appropriate volume beaker for UA.
7. Magnetic stir bars for beakers in #6.
8. 250 mL beaker wrapped with aluminum foil (or equivalent).
9. Stir plate.
10. Timer.
11. 5 M NaOH (filtered).
12. 0.22 μm filters and syringes.
2.3 Staining
Workflow
1. Parafilm.
2. Filter paper (Whatman #1).
3. Pipettes (capable of dispensing 50 μL and 2–4 μL).
4. Milli-Q water.
5. (optional) Vacuum system with a fine tip and filtered to block
the aspirated stain reaching the vacuum source.
3 Methods
Preparation of Uranyl Stains.
Important: UA and UF are light-sensitive, toxic, and radioactive. Adhere to appropriate health and safety procedures
(including use of a fume hood due to the stain powder) and
also separate stain equipment from other lab equipment.
3.1 Uranyl Acetate
(2%) (Concentration
Can be Adjusted)
Here, it is important to minimize light exposure of UA during the
following steps.
1. Weigh UA for a final 2% (w/v) and add to Milli-Q or doubledistilled water in a glass beaker with a stir bar.
2. Stir in the dark until fully dissolved.
3. Filter solution using a 0.22 μm filter and store in the dark. UA
solution can be stored at room temperature and used for
months.
Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples
119