Setting media vs non-setting mounting media: Which is right for you?

Mounting your stained specimen is typically the last step in your immunohistochemistry or immunofluorescence workflow prior to imaging (1). By the time you get to this stage of your experiment, it might be hard to think about anything but your results, but it’s worth taking the time to choose the right mounting medium (for a primer, look no further than our post Considerations for Mounting Media Selection). Here, we’ll dig into a key mounting media question: Will setting or non-setting media work best for your experiment?  

What is setting vs. non-setting mounting media? 

Mounting media can either harden into a solid film upon drying (setting) or remain in a liquid state (non-setting). Almost all solvent-based (non-aqueous) media are setting, so if you’re interested in using a non-setting media, you’ll probably need to choose a water-based (aqueous) mounting media. Both setting and non-setting formulations can provide mounting media’s main function, which is to physically protect the specimen and keep it from drying out (1). Neither is better than the other; it’s all about the right fit for your experiment. 

Can you archive your stained specimen for a long time?  

Setting media are generally better suited for long-term storage, and some allow you to keep your stained slides at room temperature. Non-setting mounting media can be a great choice if you don’t need to save your slides for very long, but remember that specimens mounted with non-setting media may require storage at 2–8°C. 

How do you prevent bubbles and shrinkage when mounting your slides? 

Shrinkage occurs with some setting media and can alter the appearance of cell morphology. It may also lead to the formation of bubbles, which tend to act like tiny lenses and scatter light. Both bubbles and shrinkage can make crisp, unambiguous visualization of your specimen more challenging. Since non-setting media remain liquid, you won’t need to worry about retraction or shrinkage. If you’d still like to use a setting media, don’t worry: Vector Laboratories offers a variety of both setting and non-setting media that will keep your specimen crystal clear.  

Can you immediately view your mounted slides under the microscope? 

If you want to take a look at your samples right after mounting, a non-setting media might be the best choice. Since these media stay in a liquid state, they don’t need to harden prior to imaging. Setting media, on the other hand, will have to dry before you image your specimen, and some require that you wait for as long as 24 hours for the best viewing results. 

How do you seal your mounted slides? 

If you opt for a non-setting mounting media, you may need to apply a sealant (like nail polish or plastic sealant) around the edge of your coverslip to keep it in place. You may enjoy giving your slides a little manicure before imaging, but this process can be time-consuming if you need to mount a large number of slides. What’s more, some sealants can cause quenching or background fluorescence (2). Consider using a setting media if you’d prefer to skip this step.  

Can you reverse coverslipping your slides? 

Are you the kind of scientist that has more ideas than you do specimens? Both setting and non-setting media might be able to help you out. It’s possible to remove the coverslip of mounted sections by soaking the slide in buffer, allowing you to re-stain your specimen and then mount again. This can also be a great trick to get rid of any unwanted bubbles that happen to pop up in your mounted samples (3).  

Conclusion 

Now that you know all about the pros and cons of setting vs. non-setting media, you might want to consider what else your mounting media can do for you. This stellar solution doesn’t just protect your specimen: It can also improve the optical clarity of your sample when you view it under the microscope or prevent your fluorescent signal from photobleaching (fading). If you’d like to make the most of all the advantages that a high-quality mounting media can offer your experiments, we have a few suggestions for you. 

• VECTASHIELD® PLUS Antifade Mounting Media is our latest new and improved mounting media with unbeatable antifade technology and absolutely no inherent toning or background in all channels. With this powerhouse non-setting formulation, you won’t need to seal your coverslips after mounting, you’ll be able to view your slides in one hour, and you can even store mounted samples for extended periods without losing signal intensity. VECTASHIELD® PLUS Antifade Mounting Media is available in two different sizes and comes with or without DAPI, so you can choose exactly what will work best for your experiment. 
• VECTASHIELD® Vibrance™ Antifade Mounting Media is another recent addition to the VECTASHIELD® Antifade Mounting Media family. It has superior anti-photobleaching performance across the entire spectrum, is compatible with commonly used fluorophores, and allows you to view your sections just one hour after mounting. Plus, VECTASHIELD® Vibrance™ Antifade Mounting Media offers minimal bubble formation and extended archiving of slides at room temperature. If you’re looking for a setting media that also offers many of the perks of non-setting media, this new hotshot solution might just be the one for you! 

Now that you know how to choose a mounting media that’ll take your staining across the finish line, there’s really only one question left: What incredible discovery will you make next? If you run into any trouble along the way, check out our SpeakEasy Science Blog and Protocols and User Guides for support with every step of your staining workflow. We’re here to help!  

1. Ravikumar S, et al. 2014. Mounting Media: An Overview. Journal of Dr. NTR University of Health Sciences.
2. The University of Arizona. 2004. Light Microscopy: Tips.
3. Bekkouche BMB, et al. 2020. Comparison of Transparency and Shrinkage During Clearing of Insect Brains Using Media With Tunable Refractive Index. Frontiers in Neuroanatomy.