Hey everyone,
We have been trying to image some amyloid fibrils under STEM using 2% UAR uranyl acetate replacement stain but we haven't been consistently getting good images. Most of our grids when we imaged them have a lot of junk, making it hard to find any fibrils.
We normally place a few uL of our sample onto carbon coated EM grids for a few minutes, remove excess liquid and add a few uL of 2% uranyl acetate replacement stain for a few seconds before washing with deionised water and letting it to air-dry for an hour.
We have been able to find some fibrils when we prepared them without adding any stain, and we did successfully find some fibrils using the method above (I've provided some images). However, that was using fibrils made out of Neurokinin B (NKB); we have been unsuccessful in producing good images of stained/unstained amyloid-beta fibrils using the method above. Additionally, when we replicated the same method, most of the grids had a large amount of junk on them; it seemed to be an one-off.
Does anyone have any tips on how to prepare fibrillar proteins using uranyl acetate replacement stain such that we can minimize the amount of junk/artefacts present on the grid?
Thanks.
You have a problem - poor focusing at higher magnifications, images are mostly useless. Either use plain TEM (could be much better for your task) or properly align your scope in STEM mode.
For negative staining you may want to try 1% phosphotungstic acid (PTA) in water instead of your substitute stain.
Hello Dr. Jayawardena,
Looking at your TEM images it is quite clear that your material is getting over-stained. This can be avoided/reduced by the following three methods:
1. Instead of using lower amount of higher concentration staining agent, it is more advisable to use higher amount of lower concentration staining agent. You can go with 0.01 (M) staining agent.
2. Also, you should disperse the solution very nicely containing your material to avoid agglomeration.
3. Lastly I presume you are not washing it off nicely. Since its an acetate salt, it may well be dissolving and staying back in the grid!
All the best!
You have a problem - poor focusing at higher magnifications, images are mostly useless. Either use plain TEM (could be much better for your task) or properly align your scope in STEM mode.
For negative staining you may want to try 1% phosphotungstic acid (PTA) in water instead of your substitute stain.
Hello Bhawantha,
It seems to me that your carbon coated EM grids are a little bit hydrophobic. Did you use some carbon support film treatment before sample preparation? If not you should try glow-discharge activation of carbon coated grids before negative staining.
Your samples are probably positively stained and not negatively stained. This might be caused by your sample preparation procedure. You wrote: “ … add a few uL of 2% uranyl acetate replacement stain for a few seconds before washing with deionised water ...”. In the classical negative staining procedure there is no washing step. Please look at the chapter of Hayat, M. A. (1986), Negative Staining, Basic Techniques for Transmission Electron Microscopy', Elsevier BV, , pp. 232-264 or at some other electron microscopy methods manual for details.
Regards
Oldrich
Osmium tetroxyde is a very powerful and useful (although highly toxic - careful!) staining in biology. It reacts mainly with C=C, but reactions with -OH, -COOH, etc are also observed. Since your fibrilar proteins have all these groups, it will stain. Moreover you can try to stain using the vapour of OsO4 solution and you will notice less staining
you can try to lower the Uranyl Acetate concentration to 0.1-0.5% - that can give you more consistent results, since you'll observe less UA crystals. You can also try to improve your washing step to get rid off the excess of UA.
Osmium tetroxyde is a very powerful and useful (although highly toxic - careful!) staining in biology. It reacts mainly with C=C, but reactions with -OH, -COOH, etc are also observed. Since your fibrilar proteins have all these groups, it will stain. Moreover you can try to stain using the vapour of OsO4 solution and you will notice less staining contamination.
But the staining is not your only problem here. As Vladimir Dusevich pointed out, you need to improve the alignment of your SEM to get nice images or use a TEM.
Good luck!
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