Prerequisites for Immunoelectron Microscopy

(http://www.hopkinsmedicine.org/micfac/FastFacts/ImmunoElectronMicroscopy.html)

You've just decided that you want to use immuno-electron microscopy (immuno-EM) to identify your
favourite protein/gene-product within cells. Before you rush to the Microscope facility, antibody and cells
in hand, make sure you are ready for this ultimate technique of locating proteins within cells. Despite the
high-quality information that it can provide, immuno-EM can be very laborious, and hence expensive to
perform. Sometimes, it just doesn't work, despite everyone's best efforts. To maximize your likelihood of
success, we provide this checklist of prerequisite information and experiments that you should read
before consulting with us about your project
Literature search: prior immuno-EM (and immunofluorescence) efforts with your tissue?
For successful immuno-EM, two things have to work at the same time. Antibody/antigen binding has to
be strong and specific, while simultaneously, ultrastructure has to be stable enough for electron
microscopy. Procedures good for one set of constraint are not necessarily good for the second set of
constraints. If someone else has developed fixation protocols of your tissue that both preserve
morphology AND preserve labelling of epitopes, you're establishing a credible starting point for efforts. If
no one has ever succeeded with your tissue, you will probably face more challenges. However, if it's
important enough, the high quality information from immuno-EM is worth the effort.
So before embarking on immuno-EM, you may wish to do a literature search for your tissue. Keep an
eye out for variations in fixation procedures, both for immuno-EM and for immunofluorescence
(particularly aldehyde fixation protocols, see below). Although not necessary, prior success in the
literature just bolsters our general confidence that something ought to work. By the way, if you think the
papers are promising, you should bring PDFs of the relevant articles to your consultation with us.
Quality of Primary Antibody
As with any immunological technique, everything hinges on the quality of your primary antibody.
Antibodies with poor specificity or poor affinity will doom your efforts. These days, everyone checks
their antibodies using Westerns, thus evaluating the specificity of the antibody. However, Western blots
immobilize proteins in their denatured state. Your antibody might be great for a denatured peptide, but be
lousy for the native protein. In a western you have denatured protein and no fix. The protein is also
concentrated into a thin band. By denaturing the protein the epitopes are generally available to the ab and
without the fix the masking of the protein is normally not a problem.
On sections though you have to make the protein available and find out what fix is compatible with the
ab's. Some fixes cross link and cover up the epitope and others can change the confirmation of the protein
also masking the epitope. Given that each protein has a unique make up you can easily see how this can
get complicated.
Hence, using the Western blot, alone, for evaluating the quality of your primary antibody is not sufficient
for fluorescence microscopy, much less immuno-electron microscopy. Better tests using
immunofluorescence are discussed in the next section. In fact, immunofluorescence is the main
prerequisite we need to evaluate the probability of successful immuno-EM.
In addition to specificity and affinity, your primary antibody preparation should be biochemically clean.
Even though we use commercial secondary antibodies bound to gold particles, secondaries may not have
enough specificity to detect your primary antibody in crude sera. Prior to attempting immuno-EM with
custom-produced antibodies (sera, ascites, or secreted monoclonal growth media), we suggest that you
affinity-purify your antibody and measure its protein concentration, if you haven't already. Commercial
primary antibodies are typically clean enough.

Immuno-fluorescence tests of antibody under EM-like fixation conditions

The best indicator of successful immuno-EM is immunofluorescence evaluation. Obviously, your first
attempt for immunofluorescence should be the conditions that others have used for immunofluorescence
of your cells/tissues. Don't forget negative controls by using pre-immune sera/antibodies you should
have no signal when using pre-immune sera/antibodies. Ideally, you have very bright immunofluorescent
localization of your primary antibody, and the background signal is very low or absent. Remember that
immuno-EM inherently produces higher apparent background labelling than immunofluorescence. Please
bring these images for your consultation. Not only do they document the specificity of the antibody, they
also provide a roadmap for guiding subsequent discussion.
After you've convinced yourself that your antibody is both specific and good signal, you are ready to
explore the conditions typically used for electron microscopy. Use the same primary antibody and the
same tissues. Although popular and successful for fluorescence microscopy, solvent fixatives, such as
methanol, acetone, or ethanol, cannot be used for electron microscopy. Because these fixatives coagulate
proteins (sub-wavelength) while extracting some lipids, they do a poor job of preserving ultrastructure. Of
course, if you don't care about ultrastructure while pursuing immuno-EM, then you don't need to explore
EM-like fixation conditions.
In general, aldehyde fixatives, such as formaldehyde and glutaraldehyde are preferred for electron
microscopy. However, for fluorescence microscopy, these fixatives have potential disadvantages.
Aldehyde fixatives, particularly glutaraldehyde, can generate significant autofluorescence that is not
related to the primary antibody. Chemical tricks, such as sodium borohydride reduction, can reduce the
autofluorescence background. Paradoxically, despite these tricks, fixative conditions good for electron
microscopy may not appear consistent with good immunofluorescence on a typical wide-field
microscope. As a last resort, the Zeiss 510 Meta laser confocal has a spectrometer that can accommodate
autofluorescence, potentially "rescuing" the antibody signal, despite the high optical background. The
empirical demonstration of feasibility is worth a lot for immuno-EM.
Please consult with us, both before and during your attempts for immunofluorescence under EM-like
fixation conditions. Typically using mixtures of formaldehyde and glutaraldehyde, we can provide you
with the recipes and protocols. Tissues require different conditions from cultured cells, and buffers can
affect the rate and quality of fixation. In consultation with you, the recipes and protocols can be tweaked
for your application, and we can provide you with the appropriate quality (EM-grade) reagents. We have
experience of chemically fixing a wide variety of samples, and we can help you navigate the trade-offs for
each decision.
Basic approaches to immuno-EM
There are five basic techniques we use for immuno-EM: (1) pre-embed labelling, (2) post-embed
labelling, (3) cryo-sectioning without embedding of lighty chemically fixed specimens (4) post-embed
labelling, after high pressure freezing (HPF) and freeze substitution (FS), (5) )cryo-sectioning without
embedding of rehydrated HPF and FS specimens. In all approaches, the immuno-labelled section must be
further stained with heavy metals (such as uranyl acetate for negative staining) to reveal ultrastructure on
the transmission electron microscope.
1. Pre-embed labelling is labelling your cells or tissue with antibodies before it is embedding in a
resin. This technique requires detergent extraction to remove or "puncture" cell membranes so
that antibodies can enter cells. Unfortunately detergents can destroy fine structure of cells and
certain antigens, particularly membrane-bound antigens, can get lost. Of course, if prior studies in
the literature have been successful with a particular permeabilization technique, pre-embed
labelling remains a viable option.
2. Post-embed labelling is labelling thin sections of cells after they are fixed and embedded in
resin. Unlike typical EM resins, this technique usually uses a hydrophilic acrylic resin that is
polymerized at low temperature, thus preserving antigenicity. Antibody incubations are performed
on grid-mounted thin sections. This technique can give you the best fine structure and

morphology because the cells are embedded in a resin. However, the trade-off is that you need an
abundant antigen. Only antigen on the surface of the section is accessible to the antibody (resin is
not permeable). In addition, some of the chemicals used to embed cells in the resins can denature
antigens, further reducing signal.
3. Cryo-sectioning, developed by K. Tokuyasu, freezes samples without embedding prior to ultrathin sectioning. As with post-embed labelling, antibody incubations are performed on gridmounted thin sections. Because samples are not embedded, the time required for resin
polymerization is unnecessary, so sample processing is faster. Because there is no resin, antigens
are likely to be more exposed and accessible to antibodies. However, the lack of plastic resin
means ultrastructure is not as well preserved in some tissue types. Again, prior studies in the
literature can give a clue about the magnitude of the issue. In the absence of other information,
our inclination is to try cryo-sectioning first for immuno-EM.

4. Post-embed labelling, after high pressure freezing and freeze substitution. HPF can preserve
cell ultrastructure and antigenicity significantly better for most cell types than conventional
methods of specimen preparation. Pressures of around 2000 bar suppress ice crystal formation
and growth and permit freezing of larger samples than ambient pressure freezing techniques. The
low-temperature substitution of dehydrating agents and fixatives (freeze-substitution) into
rapidly-frozen samples allows for the cross linking of cellular components and the removal of
water at temperatures low enough to avoid the damaging effects of ambient-temperature
dehydration. Successful cryofixation followed by freeze-substitution, shows superior preservation
of fine structure over that yielded by chemical fixation techniques. In addition, the advantage of
low-temperature embedding for immunolabelling has greatly contributed to our ability to
preserve and accurately localize antigens at high resolution.
5. It is also possible to cryosection and label HPF and FS prepared samples. This technique avoids
impregnation with resin, which some antigens are sensitive too or if the antigen is particularly
sparse.

As you can see, none of these techniques are without pitfalls. Successful immuno-EM may take multiple
attempts to develop just the right protocol. Preserving antigenicity while preserving ultrastructure is very
tricky, but we have a track record of success.