Negative Staining Protocol for Transmission Electron Microscopy

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Negative staining Protocol for Trasmission Electron Microscopy

Please read the Material Safety and Data Sheets and product description of all materials that you will be using before using any of those materials, and follow appropriate laboratory safety guidelines.

Preparing the Grids

1. Fill a large bowl (eg 25 cm in diameter) with distilled water.
2. Using a piece of microscopy lens paper, remove any dust on the surface of the bowl.
3. Place one drop of 2% parlodion (in amyl acetate) [you should be able to purchase 2% parlodion in amyl acetate] onto the water. Try not to inhale the vapour from the parlodion.
4. Wait a few minutes for the parlodion to spread.
5. Using very fine forceps suitable for handling grids, handle the grids only at their very edges and place the carbon grids (can be purchased at Ted Pella, 200 mesh size usually suitable) either light side up or dark side up onto the parlodion. Be consistent when using either light side or dark side for all the other grids. Try to place the grids not too close to the edge or the centre of the parlodion. The centre of the parlodion is generally too thick and the edge of the parlodion is generally too thin. Be careful when using the forceps because they are very sharp. Also be very careful when storing the tweezers and placing the cover back onto the tip of the tweezers.
6. Place roughly 16-20 grids reasonably closely spaced to each other in the shape of a 4x4 or 4x5 pattern.
7. Place a piece of microscopy lens paper cut to be the right size that fits over the 4x4 or 4x5 pattern.
8. With forceps, carefully cut out the parlodion section containing the grid and lens paper.
9. Air dry overnight in a petri plate, with the lid of the petri plate half open and some Whatman filter paper at the bottom of the petri plate.
10. Carbon coat the grids the next day or later on a carbon coating unit. (Ask someone to show you how to use the carbon coat unit. In general, you place the lens paper containing the grids into the vacuum chamber, lower the vacuum sufficiently, and then coat with carbon for 8-10 seconds. )
11. Rinse the bowl with only distilled water, let it dry, and put it away - use the bowl only for the purpose of preparing grids.

Negative staining

1. Cut circular Whatman filter paper into several pieces in the shape of a wedge similar to cutting a pie. Place these cut up pieces of filter paper into a petri plate. They will be used to dry off liquid on the grids.
2. When performing negative staining, try to ensure that all solutions are as fresh and clean as possible.
3. Adsorb the sample onto the grids: Using the fine forceps, pick up a carbon-coated grid face up at the very edge of the grid. Using the piece of rubber that's used to clamp the forceps together, carefully slide it up the forceps so that the forceps hold the grid securely. If the tweezers do not come with a piece of rubber to clamp it together, it may be possible to cut roughly 0.75 cm of such a piece from relatively hard rubber tubing that is the right diameter for the tweezer. Practice moving or wiggling the piece of rubber up and down on the tweezer to help loosen the rubber a bit. Be very careful that the tip of the tweezer points away from you and your hand to prevent yourself from getting poked. Place the tip of the forceps containing the grid face up with the tip of the forceps resting gently on the lid of a petri plate. Place 5 ul of the sample to be negatively stained onto the grid, and wait five minutes.
4. While waiting for the adsorption, take 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 0.5 ml for later use. (2% uranyl acetate should be stored in the fridge away from light.)
Stock solution of uranyl acetate should be weighed and stirred in the fume hood. You should heat the solution on a hot plate as it stirs to dissolve the uranyl acetate powder. You should also allow the solution of uranyl acetate to cool before removing it from the fume hood.
5. Remove the liquid with the cut up wedges of Whatman filter paper.
6. Wash twice with PBS for a minute each either by placing 5 ul of the PBS onto the grid or by inverting the grid onto 2 drops of 5 ul each of PBS applied onto a piece of parafilm.
7. Apply 5 ul of the 1% uranyl acetate onto the grid for 10 seconds, remove with Whatman filter paper, and then apply another 5 ul of the 1% uranyl acetate for 50 seconds.
8. Place the grid onto a grid holder for electron microscopy grids (can be purchased at Ted Pella).
9. Obtain training on how to use a particular transmission electron microscope and examine the samples that have been negatively stained.