Hi Everyone,
I would like to do Immuno-SEM (not TEM) on brain slices. The antibody labeling will need to be post-embedding for reasons I don't want to get into. However, I will need to omit the osmium tetroxide step during sample fixation/prep because it destroys antigenicity and also slice very thin (~70 nm) to ensure antibody penetration through the sample.
1) What are the disadvantages of omitting osmium tetroxide in brain preps (embedded in LR white). The brain is 50% lipid by dry weight
2) Are "plastic sections" (i.e. LR white embedded) suitable for SEM and are ~70 nm slices, which is more typical for TEM, suitable for SEM?.
I have never done immuno SEM and I never worked with brain, so no advises on OsO4. However, you do not need to have really thin sections. It does not matter if you have "full" antibody penetration, and I doubt that even at 70 nm you'll have it: you need relatively big gold particles for SEM (compared to TEM), which will do not go deep anyway.
I have worked with TEM-prepared grids utilizing SEM (just much faster work with SEM). Embed 812 is fine in SEM, and I believe LR white should be also OK. I worked with thin sections of mineralized tissue (bone), which were visible with BSE, but contrast, of course, is much worse compared to TEM. I expect you'll need a really good staining to see cells.
I had not bad results with observing cells in bulk specimens. I used block faces after sections were cut from them for TEM. Stained them and observed them (see attachement: osteoclast on bone surface - from "SEM in dental research"). But highest magnification I could achieve without burning embedding resin was x15,000. Do not know if it is enough for observing gold particles.
Thanks for your answers.
How big gold particles do I need for SEM? 10 nm?
I have not much experience with SEM but may be able to add some comments here.
For the immunostaining you will indeed have to omit OsO4 post-fixation. If you haven’t got your samples yet, you might want to consider to add 1% tannic acid to the formaldehyde fixative. This mordant will give some extra contrast to e.g. membranes w/o destroying epitopes.
If penetration of the Ab is a problem, there might also be solutions for that. Nanogold conjugated Fab-fragments as 2nd Ab, followed by gold- or silver enhancement.
Good luck!
Hi Norelle,
I have no immune-SEM experience, but I have examined some biological tissues by SEM and have a lot of experience in biological TEM and AFM.
Some thinks about your question:
1. It is not necessary to have the ultrathin slices (like for TEM), you can examine the classical samples for light microscopy (obviously, without a coverslip) after metal coating by standard coater for SEM. I have some experience of such samples examination. It is possible to have a very contrast and informative image in BSD mode.
2. OsO4 is not needed in this case. Usually it used for staining the ultrathin slices for TEM. The specific binding provides a distribution of "electron density" on slices for transmitting electrons. It is not necessary for examination of non ultrathin slices by SEM.
3. The very interesting way is the using of a microanalysis in mapping mode for immune-SEM. If you have the metal labeled antibodies, you can treat the slices by classical immunohistochemistry protocol, coat it by thin (about 5 nm) other metal layer by coater and examine by SEM with elements mapping. You can obtain a high quality image in BSD mode with distribution (color dots) of elements. The dots for element which is a label for antibodies will image the distribution of investigated antigen on the slices. I have examined by this method some samples of tissues contaminated of metals and had very informative results.
I hope my answer is helpful :)
Good luck!
You might consider to add Ruthenium red to your fixative. Ruthenium red added more contrast when unltrathin sections for TEM were poststained with Rr. We do not have any experience with Rr for SEM, and with immunoelectron microscopic techniques after application of Rr. Nevertheless, a trial might be useful; after all, many of the structural components of bacteria showed significantly higher contrast, including cytoplasmic membrane, envelope of PHB inclusion bodies, polyphosphate, wall layers, capsule etc. Ref.: J.Basic Microbiol. 55, issue 5, pp.349-355, 1995
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