After countless immunos with free-floating sections – troubleshooting, testing antibodies, and finally doing the actual experiments – I felt like an expert on immunohistochemistry. I knew everything there is to know, right? Well, of course not – it does not work like this in science! For my next project, I would need to perform immunohistochemistry on slide-mounted sections. Should it just be the same process, only this time on slides, or did I have to change everything in my protocol?
You, too, may be experiencing this (or the reverse) problem. Or maybe you are just trying to figure out the best method for your study. What are the differences between the two methods, and when should you use them? Below, I share some of what I learned from using them both.
Free-floating immunohistochemistry is performed with the sections floating in solution, typically in a well plate. The sections are not mounted on slides until after completing the immunohistochemistry process. In contrast, slide-mounted immunohistochemistry is performed on tissue that is already adhered to slides – usually during the sectioning process. All steps of the immunohistochemistry process are carried out on the slides. Both methods work fundamentally the same way and require the same basic processes, so to transition from one to the other will require more handling / technical changes than a complete protocol change.
Given the similarities between the free-floating or slide-mounted methods, it can be tempting to ask which one is better, but the question would be a somewhat misguided, as the answer depends on your experiment – the tissue you are using, and what you plan to do with it.
One of the biggest differences between the two approaches comes before you even start the immuno, with how you prepare the tissue. For slide-mounted immunohistochemistry, you typically freeze the tissue before sectioning. This allows for the thin sections necessary for adequate antibody penetration, which I talk more about below. Free-floating immunohistochemistry however, is ideal for fixed tissue. It can be used with vibratome sections to avoid potential damage that can arise during the freezing, storage, and thawing of frozen tissue. Fixed tissue can be cryprotected, frozen, and cryosectioned, and frozen tissue can be sectioned, fixed, and stained free-floating. However, the goal is to prepare the tissue with the minimal amount of processing needed for your technique. Preparation of the tissue should be part of your consideration when determining the best method. Let u’s look at each method in more detail, examining what other factors there are to consider.
Use slide-mounted immunohistochemistry for experiments aiming at examining the fine details in tissue, such as individual cell staining and fibers. These sections can be (and have to be) very thin. They can be very thin since they require much less handling than free-floating sections, as they are immediately adhered to slides. However, after mounting your sections to slides, the antibody can only penetrate from one side. This means the tissue must be very thin to allow for adequate antibody penetration.
Pros: Not needing tissue sections to float in solution can be an advantage – you can use much less solution. Take advantage of liquid surface tension to only cover the tissue on the slide – using a hydrophobic pen will greatly help in restricting the spread of your antibody solution.
Cons: Don’t assume that the antibody concentration you have used for free-floating immunohistochemistry will also work for slide-mounted (or vice versa). Since the slide results in single-sided penetration, you may need to use a higher concentration of antibody. This will depend on your antibody and will require testing to determine the best concentration to use.
Things to consider: When using a smaller amount of solution, you run the risk of evaporation and sections drying out, particularly for steps that sit overnight or longer. To prevent this, perform the incubation steps in a moisture chamber. These can be purchased, but you can easily put together a DIY version using supplies from your lab.
Free-floating immunohistochemistry can be used with thicker sections, making it a more practical method for looking at the distribution of staining through an entire brain region, or imaging structures through the depth of a section. And without the constraint of being adhered to a slide, it can be used for applications other than light microscopy. For example, free-floating immunohistochemistry is commonly used for immunoelectron microscopy.
Pros: The double-sided penetration with free-floating sections means that sections can be thicker, and that some antibody concentrations can be reduced. Keep in mind, of course, that there is only so much that the antibodies can penetrate – usually no more than 20?m per side, depending on the tissue type, incubation time and detergent concentration used. Although these sections tend to undergo more handling by the researcher, free-floating sections can maintain great structural integrity and produce vibrant staining.
Cons: Free-floating immunohistochemistry can be tough with cryo-sectioned or delicate tissue. If the tissue has not been well preserved or properly sectioned, keeping the tissue intact during all of the steps can be a nightmare. Help yourself out by learning best preservation and sectioning techniques before starting your experiments.
Things to consider: Transferring the tissue to a clean well for each new solution is ideal for a high quality immuno. The downside is that it requires a lot of handling. There are methods that can reduce the amount of tissue handling – you can leave the tissue in the same well and change solutions using a pipette, or transfer sections using well baskets. These variations come with tradeoffs. Changing solutions with a pipette will inevitably leave residue, and baskets often require more solution in a well and can result in grid mark-patterned staining on the tissue. Figure out which method works best for your experiment to maximize resources and quality of staining while minimizing damage to the tissue.
When you are not sure which method to use, first think about all of the factors discussed above, then determine how the tissue should be prepared and what thickness to use for sectioning. The method you choose should allow for adequate antibody penetration and should fit with how you plan to use the tissue after the immunohistochemistry processing.