Although many different immunostaining techniques have been developed since antibodies were first used as research reagents, it still holds true that no single method meets every need. Instead, a suitable immunostaining technique should be chosen based on the type of information required, as well as the nature and availability of sample material. Here, we compare some popular immunostaining techniques and provide advice for getting the best out of them.
Choose a technique to match your end goal
“The type of immunostaining experiment you want to perform likely depends upon the data you need to obtain,” says Virginia Bain, scientist (immunofluorescence) at CST. “If you wish to assess global protein levels in a few samples, consider western blot, whereas if you need to generate data quickly to examine one target in many samples, ELISA is a good choice. Flow will provide robust results if you want to study the population dynamics of cells, while immunohistochemistry (IHC) and immunocytochemistry (ICC) are more fitting if spatial context is required.”
For most scientific questions concerning analytes in solution, a classical ELISA based on a standard colorimetric substrate such as TMB is hard to beat, according to Dr. Tobias Polifke, co-founder and managing director at CANDOR Bioscience. “Not only is ELISA able to handle all risks of interference, but it is also the easiest immunoassay technique to optimize,” he says. “Furthermore, provided the assay chemistry and antibodies are well-chosen and antiquated reagents such as PBS-T/BSA or milk powder are avoided, home-brewed assays stand a good chance of producing reliable results with no requirement for specific assay kits from platform suppliers; this can make a classical ELISA outstandingly cost-effective.”
In situations where there is a need to maximize the amount of data acquired from an individual sample, techniques that allow for multiplexing are a sensible choice. “Being able to detect, visualize, and quantify multiple proteins at the same time is invaluable when sample material is limited,” reports Antonella Galli, Ph.D., head of characterization at Abcam. “Flow cytometry, IHC, and ICC are the preferred methods for multiplexing experiments. Depending on the chosen methodology, it is possible to study more than 70 analytes in parallel in a single sample, for example using FirePlex® immunoassay panels for flow cytometry.”
Consider simplifying your workflow with direct immunostaining
While immunoassays have historically relied on labeled secondary antibodies for detection, it has become common practice for researchers to perform direct immunostaining. “By enabling immunoassays with fewer working steps, direct immunostaining greatly reduces the potential for mistakes and makes assay optimization easier,” notes Polifke. “Yet despite these advantages, many researchers continue to argue that the signal enhancement provided by indirect techniques like biotin-avidin amplification improves assay performance. Actually, for most assays, this is a misunderstanding since signal enhancement is almost always accompanied by a concomitant increase in background noise. A more straightforward approach is to use surface blockers and assay diluents designed for interference-free immunostaining, whereby noise is lowered normally to make signal enhancement by indirect methods unnecessary.”
As well as simplifying workflows, direct immunostaining removes the need to deal with antibody host-based dynamics. Bain notes that this is especially advantageous in high-plex flow, where organizing the desired primary and secondary antibody combinations can be challenging. However, she warns that several technical hurdles are difficult for direct immunostaining techniques to overcome, including reduced flexibility in experimental design. “Where multiple antibody-fluorophore conjugates are not available for each target, you may need to commit to looking at certain targets with fixed spectra,” she says. “Additionally, because the signal obtained from a fluorophore-conjugated antibody is generally weaker compared to indirect immunostaining, it may be necessary to reserve brighter fluorophores for less abundant analytes; low signal can be particularly problematic if you’re working in a noisy sample like tissue. Finally, if you are attempting to conjugate the antibody yourself, you should be aware that challenges can arise from the act of conjugation itself, like altered antibody performance due to the addition of excess dyes or simply the incompatibility of certain antibodies with the conjugation procedure.”
Take steps to ensure your immunoassay goes to plan
Irrespective of your chosen immunostaining technique, several key factors underpin its likelihood of success. Paramount among these is the choice of antibody reagents since antibodies form the core of any immunostaining method. “Although it can be tempting to believe that an antibody will work simply because it is commercially available, this could lead to inaccurate conclusions and retracted publications,” cautions Bain. “If suitable data are not available for your desired technique, you should generate these yourself, as it is critical that antibodies are validated in each application where they will be used. Generally, a set of complementary assays (including non-antibody-based techniques) should be employed for validation work since no single assay is sufficient to verify antibody performance in a given application.”
Image: Antibody validation using a binary model. Immunofluorescent analysis of MKN-45 cells (left, positive) or PANC-1 cells (right, negative) using Calretinin (E7R6O) Rabbit mAb. Image provided by CST.
Galli also highlights the value of confirming antibody validation data using an independent immunostaining method or a complementary approach. “Data at the protein level can be confirmed by evaluating mRNA expression,” she says, “and it is increasingly commonplace for IHC and in situ hybridization to be coupled for data validation. Completely or significantly reducing signal following gene editing or RNA interference (KO/KD) of the target protein can also help to ensure reliable data. You can save considerable time by selecting antibodies that have passed a manufacturer’s in-house validation for specificity using KO cell lines as a suitable negative control.”
Polifke stresses that for any immunostaining technique, minimizing interference is critical. “Interference occurs when antibodies or analytes interact with binding partners additional to each other,” he says. “While this basic biochemistry cannot be changed, modern surface blockers and assay diluents are designed to lower unwanted cross-reactivities and other forms of interference. Indeed, by replacing a BSA-based blocking solution with an advanced blocker, we’ve been able to reduce immunoassay CVs from 11% down to just 3%.”
Lastly, it is essential to confirm any immunostaining data is trustworthy by including relevant controls in your experiment. “If possible, work with multiple antigens and confirm that you generate the same result with each,” suggests Bain. “It is also recommended that you include negative controls like omission of the primary antibody or a concentration-matched isotype control, and positive controls such as housekeeping gene proteins, as well as treatment controls where applicable, to increase confidence in your results.”