Principles & Protocols

Protocols - Negative Staining

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You will need:

• Gloves
• Parafilm
• Tweezers
• Aqueous Uranyl Acetate, PTA, or whichever stain is to be used
• Carbon Coated Grid or PlasticCoatedGrid.
• Pipette
• Filter paper

Procedure:

• Glow Discharge grids
• Put on gloves!
  • Most negative stains are very toxic, especially Uranium salts.
• Secure the grid in a pair of tweezers
• Pipette sample (~3 microliters) onto glow-discharged grid.
• After about 30 seconds, blot the excess sample from the grid with filter paper
• Place a drop of UA aqueous on the grid
• Blot with filter paper immediately
• Repeat the drop of UA aq and blotting a total of three times

Protocols - Deep Stain

• 10% Trehalose
• 5% bacitracin

Procedure:

• Glow Discharge grids
• Put on gloves!
  • Most negative stains are very toxic, especially Uranium salts.
• Take 20ul of your chosen stain and add 2ul of trehalose and 2ul of bacitracin
• Secure the grid in a pair of tweezers
• Pipette sample (~3 microliters) onto glow-discharged grid.
• After about 30 seconds, blot the excess sample from the grid with filter paper
• Place a drop of the stain solution on the grid
• Blot with filter paper immediately
• Repeat the drop of stain and blotting a total of three times
• Add a fourth drop of stain, and wick it from the edge with the tweezers, leaving the filter paper until the drop does not grow anymore
• At this point, shake the grid vigorously to dry it as fast as possible

What to look for:

• This procedure produces regions in which a significant amount of stain remains on the grid. In these regions, the particles can be completely embedded in a slab of stain and have excellent structural preservation. Aim to look for regions that appear like rivers, and take pictures at the edge of these, where the stain is deep enough to cover the particles, but not too thick.

• These samples can also be prepared over small holes, in which case the stain film spans the hole and gives the same appearance of cryo negative staining.

• Set ALLOWTOPICCHANGE = CemfacGroup

-- DavidStokes - 18 Mar 2005