Mounting Media

What are mounting media?

Mounting medium is the medium that your sample is in while it is being imaged on the microscope. The simplest type of mounting medium is air, or a saline-based buffered solution, such as PBS. Because most people use the term mounting medium when referring to fixed-cell imaging performed with immunofluorescence labeling, during live-cell imaging, the term imaging medium is more often used to refer to the medium that samples are in while they are being imaged.

There are several reasons to place your fixed-cell sample in a mounting medium while you image:

• To help hold a specimen in place while you are imaging
• To prevent your sample from drying out
• To more closely match the refractive index for the objective you will use
• To prevent photobleaching
• To preserve your sample over time for long-term storage

The choice of mounting medium is largely dependent on your sample type, how you will image, and which fluorophore or fluorescent proteins you use. There is a wide variety of mounting media to choose from, whether you buy commercially available versions or want to “brew” your own, and they can differ widely in composition. Some are based on organic solvents such as toluene or xylene, others are water-based or aqueous mounting media.

Mounting media for fixed-cell imaging

There are two main types of mounting media: water-based and solvent-based. The main difference in workflow is that for aqueous mounting medium, samples can be directly transferred from buffer to the mounting medium. In contrast, when you use a solvent-based mounting medium, your sample will usually need to undergo dehydration steps (in solvent or air) prior to transferring it to the mounting medium. These solvent-based mounting media can preserve the sample the longest.

The most common type of aqueous mounting media is simply a buffered saline solution such as PBS. Imaging in buffered saline solutions provides the opportunity to quickly image samples with minimal postprocessing steps after antibody labeling and staining, and allows you to quickly check your sample to see if the staining looks like you expect. This is nice if you want to label your sample with multiple fluorophores and need to check your staining progress at each step, or if you aren’t sure your protocol will work and want an answer right away.

Some types of mounting media can help protect and preserve samples

Formulations of mounting media that can add favorable properties such as optimizing the refractive index to match that of glass, preventing photobleaching, or preserving samples for long-term storage are widely available. Keep in mind that some require time to “cure” or harden. For mounting media that need time to cure, it’s important to let the sample fully harden before imaging so that you don’t inadvertently damage or destroy your sample by moving it around on the slide or cause photobleaching of your stained cells. Typically, the refractive index of the mounting medium will not reach its specified value until after it has fully cured, and its photoprotective properties will increase during the curing process. The degree of hardness after the mounting medium has fully cured will also vary; some will set like jelly while others will set like hard plastic.

Sealing liquid-mounted samples can help prevent drying

When using PBS or other liquid mountant, you may decide you want to seal the coverslip edges to prevent the sample from drying out or to preserve it better for longer-term use. Typical sealants such as melted paraffin wax or fingernail polish are painted along the edges of the coverslip to serve as a moisture barrier and to help adhere the coverslip to the slide. If you choose to seal your coverslip, be aware that some sealants are themselves fluorescent, and you will want to avoid imaging any areas that are close to the sealant so that you don’t have to worry about this extra background fluorescence.