Tips for Immunofluorescence Microscopy

Immunofluorescence microscopy (also known as IF microscopy) is a very useful technique for the detection and localization of cellular proteins and other antigens via fluorescent-labeled antibodies. By revealing the spatial distribution of antigens in the cells, immunofluorescent labels coupled to highly specific antibodies provide information on the dynamics of protein trafficking between cell compartments as well as between the cell and the extracellular space. In most cases, cells are fixed and permeabilized, blocked, and successively incubated with a primary and a fluorescently labeled secondary antibody. Despite being a straightforward procedure, several variables significantly contribute to successfully achieving remarkable staining patterns and thereby publication-quality images. Continue reading below to learn more on how to properly optimize your IF experiments.

• Cell Fixation and Permeabilization

  To guarantee optimal antibody detection, cells need to be properly fixed and permeabilized. These are critical, cell-dependent parameters that need to be balanced for antibodies to reach their targets while maintaining cell and antigen integrity with as much fine structure preserved as possible. In general, you will find that at least one of the following should accomplish this goal: cell fixation with 2%–4% paraformaldehyde followed by permeabilization with one of the following detergents: 0.1% saponin or 0.3% Triton X-100. Usually the former is a more mild treatment but might be ineffective with nuclear targets which are better reached when using Triton. When using saponin, be aware that contrary to Triton, it causes only reversible permeabilization of the cell membrane, meaning you’ll have to include it not only during the initial permeabilization step but with each antibody incubation. Alternatively, cells can be simultaneously fixed and permeabilized with ice-cold methanol, circumventing the use of detergents at all.

• Antibody Specificity

  Perform IF only with antibodies that show high specificity for the antigen of interest. This will reduce high background and unreliable patterns of protein localization. Affinity-purified antibodies should work well in most cases but sometimes even affinity-purified antibodies exhibit more than a single reactivity in cell lysates. Including the right controls will always help to determine the specificity of the antigen being recognized. Always use slides stained only with a secondary antibody to determine the threshold of background signal and also include cells lacking the antigen of interest whenever possible. The use of an isotype of the primary antibody is also suitable as a control. If working with anti-serum, a useful control is including cells stained with pre-immune serum.

• Using the Appropriate Antibody Dilution

  It is possible to improve staining by adjusting the antibody dilution. Usually, 1 µg/mL of purified antibody or 1:100–1:1000 of anti-serum should be enough to achieve specific staining. It is always possible to enhance the intensity of the signal as long as the background remains low. If it is the first time you are either using an antibody or characterizing an antigen, it is highly advisable to titer the reagents through a series of dilutions.

• Optimizing Buffers and Blocking Agents

  Although most antigens will stain well in common buffers like PBS, a significant improvement in few targets might be achieved by switching to buffers with different compositions of ions like calcium, magnesium, and potassium. A variety of blocking agents have also been successfully used in IF but 5–10% fetal bovine serum is, in general, a good start. Other blocking agents like BSA fraction V, gelatin, serum from the same species as the secondary antibody, or our optimized blocking buffer for IHC can also be used.

• Using an Appropriate Cell Density

  Choose several cells that will result in about 50% cell confluence at the time of staining. When the cell number is excessive, the cell architecture might not be well appreciated and may result in higher background at low magnifications. Low cell numbers will reduce the probability of finding a field with the optimal pattern. Also, because non-adherent and weakly-attached cells might be tricky to grow on glass surfaces, they can be attracted into adhering by coating the coverslips (12 mm, thickness #1) with poly-lysine or extracellular matrices like collagen or laminin.

• Multiple Staining

  When the conditions for successful staining have been established independently, it is possible to study the expression and co-localization of two different antigens in the same sample. This can be done by incubating them with their corresponding antibodies simultaneously. In this case, each primary antibody should be generated in different species to use fluorophore-conjugated secondary antibodies that can be detected by separate channels. These fluorophores can range from small molecules like DyLight, ATTO, or Cy to bigger proteins, such as RPE. Alternatively, samples can be sequentially double-stained, in which case all blocking and primary and secondary incubations are completed first for one antigen and then performed for the second one. In general, the sequential approach seems to generate better images and fewer artifacts.

• Secondary Antibodies

  You should use pre-adsorbed secondary antibodies when performing IF. This is not only mandatory when performing multiple/double staining but highly recommended with single-antigen staining. It is also preferable that you work with secondary antibodies from the same species.

• Reducing Background

  High background may be problematic during IF. Different ways to circumvent this issue include blocking with serum (from the same species of the secondary antibody) instead of BSA, reducing the amount of antibody (especially secondary) and/or reducing incubation times, and having at least three, 5-minute washes (PBS + 0.05% Tween, recommended) between incubation steps.

• Mounting

  The final step of the IF method typically involves treating the sample with mounting solutions. This step is necessary since it improves the refractive index and helps to preserve the sample. Nevertheless, applying the right amount of mounting media can be tricky—applying too much can overflow and blemish the edge of the coverslip, whereas too little can result in air bubbles trapped between the coverslip and the slide. An optimal amount of mounting media should take about 30 seconds to spread out when the coverslip is placed on the slide.

• Data Interpretation

  Always include your secondary antibody only as well as isotype controls and other negative controls where the antigen is not present so that you can differentiate artifacts from autofluorescence and improper permeabilization, which could lead to poor staining and even misleading patterns. More importantly, since IF results are usually a picture depicting few cells, make sure the picture truly represents the more common, representative pattern observed along the whole sample.