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Immuno-Electron Microscopy Prerequisites

Great! You've just decided that you want to use immuno-electron microscopy (immuno-EM) to identify your favorite 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 laborous, 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 educational checklist of prerequisite information and experiments that you should read before consulting with us about your project.

This document is purely educational, describing an ideal progression of events. Even if you haven't executed everything or are having difficulties, it's still a good idea to consult with us about your project. for an appointment. We have a lot of experience and may be able to guide you through potential challenges.

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As you discover below, immuno-EM is very empirical, almost an art. 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 preseve 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.

Hence, before embarking on immuno-EM, you may wish to do a literature search (PubMed, for example) 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 for details). 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.

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. Cross-reactive denatured epitopes are surprisingly prevalent in cells. 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.

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 LSM780 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 many years of experience chemically fixing a wide variety of samples, and we can help you navigate the trade-offs for each decision (but there's no substitute for empirical proof!). Make us part of your research effort; just ask us!

There are three basic techniques we use for immuno-EM: (1) pre-embed labelling, (2) post-embed labelling, or (3) cryo-sectioning without embedding. 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 labeling is labeling 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 labeling is labeling 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 permeant). 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 ultra-thin sectioning. As with post-embed labelling, antibody incubations are performed on grid-mounted 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.

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 long track record of success. With this primer, you will be able to discuss your options when you consult with us. to make an appointment.

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