Thermo Fisher Scientific
Technical Applications Scientist
Tips for fixed cell imaging
Are you ready to create images from your immunofluorescence experiments that are eye-popping and publishable? Understanding the basic steps for immunofluorescence protocols will help you generate an image that best communicates your data, and these tips can make that image into a work of art.
Unfortunately, there isn’t one perfect protocol for staining fixed cells, so optimizing a protocol for your new antibody will be required. Finding information on what conditions work well with your antibody-antigen combination could help a lot in the long run. A little upfront literature search is worth the time it saves getting to that jaw-dropping image.
There are six basic steps for staining cells for imaging: sample preparation, fixation, permeabilization, blocking, antibody incubation, and mounting. Here are some tips that could help optimize your results for each step.
1. Sample preparation
Incubation conditions with the correct media, serum, temperature, and CO2 concentration are a must. Also keep in mind the substrate you are growing them on and how that will work for your imaging platform. Commonly, this means growing cells on coated coverslips in dishes or plates, but it could also mean growth in multi-well plates, in flasks in suspension, or in 3D cell culture. Upright microscopes often require cells on coverslips, while inverted systems can also image cells in multi-well plates or dishes.
Before beginning your immunofluorescence protocol, always check the cells for general health and confluency. Do you have enough cells? Do they have the expected morphology, or are they blebbing, rounding up, or coming dis-attached from the substrate?
Tip #1 – Know that your cells express the antigen for your antibody before starting your optimization for imaging. This seems straightforward but you’d be surprised how many researchers forget to do a simple western blot before jumping to immunofluorescence staining for their antibody-antigen combination.
Tip #2 – Optimize the buffers. One of the often overlooked areas of optimization is buffer selection. The buffers are used during incubations and washes after fixation and permeabilization; using the right buffer can make a big difference to signal intensity. A majority of scientists automatically turn to PBS for their staining needs, but buffers such as PHEM or CSK, which are often used at a slightly lower pH, may be helpful with cytoskeletal or cytoplasmic antigens.
2. Fixation.
Keeping cells as intact as possible through all those incubations and washes is both tricky and extremely important. Fixation is a critical step in maintaining the cellular structure. Many commercially-sold primary antibodies have been validated with a particular fixative, but a new antibody will need to have an optimized protocol established.
There are two classes of fixatives to choose from: aldehyde fixatives and organic solvents. Aldehyde fixation crosslinks proteins to one another within the cytoskeleton and membranes and is therefore the method of choice for labeling of antigens in those structures. However, this method modifies proteins chemically and can mask them, making detection more difficult. A good starting point is the formaldehyde-based Image-iT® Fixation/Permeabilization Kit, which works for most cell types. Organic solvents, such as methanol, ethanol and acetone, don’t alter the protein by crosslinking, but they do denature and precipitate proteins. This method might be good for those antibodies that have a "buried" antigen, such as in the nucleus.
Tip #3– Do not use organic solvents for fixing cells containing a fluorescent protein tag. This could denature your labeled fusion protein leaving you with no signal.
Tip #4 – Counter-act fixative-induced autofluorescence. Some samples are prone to fixative-induced autofluorescence, which can be lessened by reducing the aldehyde groups with BH4 or NH3Cl at 10 mM. Glutaraldehyde is particularly bad for autofluorescence.
Tip #5 -- Antigen retrieval methods may be needed. If an antigen is masked, such as by aldehyde crosslinking, it can often be unmasked by treatment with heat, pressure, and/or special buffers, such as a citrate buffer. Numerous reviews of these methods have been published, and sometimes commercial antibody sources will recommend a method.
3. Permeabilization
Permeabilization helps get the antibodies past the cell membrane and inside your fixed cells by delipidizing the membrane. There are a number of different detergents that can be used to permeabilize a cell, including NP-40, Triton X-100, Tween-20 and Saponin.
The extent of permeabilization needed depends on the location of your antigen and the depth of penetrance needed. For example, if your protein of interest is on the cell surface or in the extra cellular matrix, then you probably don’t need much permeabilization, if any. In fact, you could lose your membrane-bound protein by removing the membrane through this procedure. Intercellular targets, however, will require some method of permeabilization in order for the antibody to reach the antigen. Optimization of time and concentration is needed, but a good starting point is the TritonX-100-based Image-iT® Fixation/Permeabilization Kit.
Tip #6– Optimize your fixation and permeabilization methods. Do this by trying the different compounds at different times and concentrations. You are looking for good cell or tissue morphology and a strong, specific stain. A literature search can sometimes prove helpful, as well.
4. Blocking
Any immunofluorescence staining will be improved by blocking non-specific binding of your antibody to your sample due to non-specific protein binding. Bovine serum albumin and heat-inactivated normal serum can be used as non-specific blocking agents, and there are also some commercial products available for this purpose, such as BlockAid™ Blocking Solution. The blocking solution is applied prior to the antibody incubation as well as in the primary antibody incubation solution.
Tip #7– Choose your blocking agent carefully. Don’t use serum from the same species as the one in which the primary antibody was created. Nothing will get rid of that background!
Tip #8– Additional blocking solutions may be needed. Non-specific binding due to dye charge can be blocked using Image-iT FX Signal Enhancer. Specialized methods, such as enzymatic or biotin-avidin amplification will also require blocking of endogenous peroxidases, phosphatases, or biotin commonly found in some cells and tissues.
5. Antibody Incubation
The actual detection step involves bathing the cells or tissue with the primary antibody and a dye-conjugated secondary antibody. Again, final concentration and incubation time have to be carefully optimized, but usually fall in the range of 0.5-10 µg/mL for 30-60 minutes at room temperature, for microscopy of cultured cells. Multiple primary antibodies can also be combined in one step.
The primary antibody is usually detected with a dye-conjugated secondary antibody against the host species of the primary antibody. When combining multiple primary antibodies, make sure the different secondaries are cross-adsorbed against the other species or they will cross-label, leading to confusion in the signals. Make sure the dyes that are used match the filter options and/or laser lines available with your microscope and are dyes that have a reputation for being bright and photostable, such as Alexa Fluor dyes.
Tip #9– Use the best primary antibody for your target. This is one of the most important parts of the process. If this is done well, you will get a bright, specific stain ready for publication. The perfect antibody will have good affinity and specificity. It’s worth researching and paying for the best antibody available. If generating your own, take the time with designing the antibody. It will pay off in the long run. Make sure it isn't old and degraded!
Tip #10– Use appropriate controls. You will want a no-primary (secondary-only) control to test for the specificity of your secondary antibody. A control with no antibodies or dyes will also test for autofluorescence. If using multiple primary antibodies, perform single-antibody controls to test for cross-labeling.
5. Mounting
What's the point in going through all those steps and optimizations if your dye isn't stable afterward? Once the final wash is through, the labeled sample will need to be in an imaging environment that best preserves the dye signal. Though cells can be imaged in buffer or in buffer/glycerol solution, the best option is to put the cells in an antifade mounting medium which will preserve the initial signal and reduce photobleaching (dimming due to light exposure) and have a refractive index high enough to achieve excellent resolution.
Tip #11– Choose an antifade mountant which best suits your imaging timeline. If you are imaging right away and then discarding the slides, your mountant can stay liquid, such as SlowFade(R) Diamond. If, however, you wish to archive the slide for a day or more and image later, you will want to use a mountant that hardens, or "cures," such as ProLong(R) Diamond.
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