Negative Staining Protocol for Transmission Electron Microscopy

Please read the Material Safety and Data Sheets and product descriptions of all the materials you will use before using such materials, and laboratory safety guidelines accordingly.

preparing Grids

  1. Fill a large bowl (eg 25 cm) with distilled water.
  2. Using a microscope lens sheet, remove dust on the surface of the bowl.
  3. Place one drop of 2% parlodion (in amyl acetate) [You should be able to buy 2% parlodion in amyl acetate] to water. Try not to inhale the vapors from the parlodion.
  4. Wait a few minutes for parlodion to spread.
  5. Using very fine forceps suited to handle the grid, the grid handles only on the very edges and put the carbon grid (can be purchased at Ted Pella, 200 mesh size is usually suitable) for both the light side or the dark side over to the parlodion. Consistent when using either the light side or dark side to all the other grids. Try to put the grid is not too close to the edge or center of the parlodion. Parlodion center generally too thick and the edges parlodion generally too thin. Be careful when using pliers because they are very sharp. Also very careful when saving a pair of tweezers and put the cover back into tip tweezers.
  6. Place approximately 16-20 grid spaced closely enough with each other in the form of 4×4 or 4×5 pattern.
  7. Place a piece of paper into a microscope lens right size suitable for 4×4 or 4×5 pattern.
  8. With pliers, carefully cut parlodion section that contains the grid and lens paper.
  9. Air dry overnight in a petri dish, with the lid half open petri dishes and some Whatman filter paper at the bottom of a petri dish.
  10. Carbon grid coat the next day or later in the carbon coating unit. (Ask someone to show you how to use the unit carbon coat. In general, you put the lens paper containing the grid into a vacuum chamber, vacuum lowered sufficiently, and then coat with carbon for 8-10 seconds.)
  11. Rinse the bowl with only distilled water, let dry, and store it – use a bowl just for the purpose of preparing the grid.

negative staining

  1. Cut a circular Whatman filter paper into several pieces in the form similar to the cutting of the cake slices. Place the cut up pieces of filter paper in a petri dish. They will be used to drain fluid in the grid.
  2. When performing negative staining, try to ensure that all the solutions are as fresh and clean as possible.
  3. absorbing the sample to the grid: Using a good pair of pliers, take the box of carbon-coated face up on the edge of the grid. Using a piece of rubber that is used to clamp pliers together, carefully slide it upward so that the pliers holding forceps secure grid. If tweezers are not coming up with a piece of rubber to clamp together, it is possible to cut approximately 0.75 cm of a piece like that of a relatively hard rubber pipe diameter is appropriate for the brace. Practice moving or swaying rubber pieces up and down on a rubber brace to help loosen slightly. Be careful that the end of the brace that points away from you and your hands to prevent yourself from getting pierced. Place the tip of the forceps containing a grid face with the tip forceps gently resting on the lid of a petri dish. Points 5 ul stained samples to be negative to the grid, and wait five minutes.
  4. While waiting for adsorption, taking 0.5 ml of 2% uranyl acetate (in water), and dilute in 0.5 ml of water. Spin down at 4 degrees C for 5 minutes. Remove the top of 0.5 ml for later use. (2% uranyl acetate should be stored in the refrigerator away from the light.)
    a stock solution of uranyl acetate should be weighed and stirred in a fume hood. You have to heat the solution on a hot plate like stir to dissolve powdered uranyl acetate. You should also allow the solution of uranyl acetate cool before removing it from the fume hood.

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