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.

Investigators are leveraging multiplex immunohistochemistry (IHC) to tackle new research frontiers. From developing personalized diagnostics for melanoma patients to teasing out the immune microenvironment in colon cancer, the simultaneous visualization of multiple targets on the same specimen can help solve an incredible array of scientific questions (1,2). Curious about where this versatile technique could take your science? You’re in the right place! Keep reading for some Vector Laboratories-approved insider knowledge to make your multiple-antigen staining sparkle. For even more hot tips and tricks, check out Dr Craig Pow’s full-length webinar, “Multiplex Immunohistochemstry — Expand Your Insights, Improve Your Results”, and our other posts. 

Start weak to finish strong  

First things first: once you have carefully picked out your antibodies and enzyme detection systems (for advice on how to choose wisely, we have a blog post on getting started with multiplexing IHC), in what order should you apply them to your specimen? Throwing them on willy-nilly can undo all your meticulous planning, so allow us to share a few wise words from Vector Laboratories’ multiplex maven Craig, who earned his PhD in Physiology from the University of Queensland. He suggests always labeling the less prevalent antigen first and pairing it with the most sensitive detection system. Then, follow up with your second label for the more strongly expressed target. Of course, if you have more than two labels, you would stain them in order from the least to most abundant antigen. Does combining multiple substrates and figuring out how best to apply them leave you scratching your head? We put together a susbstrate selection page that makes it a breeze. 

Be a label control freak  

Next up, controls. You will need both positive and negative single-label controls for each target antigen and corresponding detection system (“The Curious Case of the Staining that Went Wrong” is a great refresher on running stellar single-stain controls). Run them in parallel—still as single labels—on separate test specimens. You will really understand where you should be seeing positive and negative results on your multiplex specimen. This will give you confidence that you’re seeing a true multiple staining result in your multiplex stain. Craig recommends running single-label stains in parallel each time that you perform multiplex immunohistochemistry. Otherwise, you may be unsure of whether you’re looking at a novel observation or a pesky false result! 

You have yielded negative controls to find out how stained tissue that doesn’t express your antigen of interest looks. Now, you will use them to sniff out any potential false-positive staining caused by cross-reactivity between your multiple labels. Start by running your first stain all the way through, including first primary antibody, first secondary, and first enzyme substrate. If it looks great, continue with your second stain, but substitute a non-immune IgG for your second primary antibody (non-immune IgG + second secondary antibody + second enzyme substrate). If you see a signal from your second label with the non-immune IgG, it could mean that either your second secondary antibody is binding to excess first primary antibody or that the second enzyme substrate is reacting with residual enzyme—this can happen if you use the same enzyme-based detection system for multiple stains, like two peroxidase-based systems. To figure out what’s going on, run your first stain all the way through again and then apply the second enzyme substrate alone. Still getting a signal? Consider adding a step to quench residual enzyme activity between your first and second stains. For workflows that call for a tertiary label, simply repeat the process with your third stain.  

Using a two-step methodology or an avidin-biotin complex kit? The same principles apply. Just as with the previous example, start by applying your first label, including first primary antibody, first secondary antibody, the biotinylated secondary avidin/streptavidin-based enzyme system, and first substrate. Then, run your second stain to completion, substituting a non-immune IgG for your second primary antibody as before. If you see a signal with the non-immune IgG, eliminate each step of your protocol systematically: the second secondary antibody, then the second biotinylated secondary avidin/streptavidin-based enzyme system, and finally the second substrate. Once you have worked out where the trouble lies and resolved the issue, you will feel confident about your staining. 

Just say no to inherent enzyme activity 

You have done all your controls, great! But what if your perfect staining is still being marred by inherent enzyme activity? Craig explains that this is particularly likely to happen with peroxidase-based detection systems and that some researchers circumvent the issue by using only alkaline phosphatase-based detection systems. However, there are many reasons to choose one detection system over another, and we would hate to see you limited by inherent enzyme activity. Instead, quench the endogenous enzyme activity with a blocking solution. Consider simplifying your workflow with a universal block like BLOXALL^® Endogenous Blocking Solution, which knocks out inherent peroxidase and alkaline phosphatase enzyme activity in one fell swoop and is compatible with all primary antibodies.  

To counterstain or not to counterstain?  

Your thoughts may turn to counterstains as you approach the end of your staining protocol, but Craig cautions that unless used carefully, a counterstain can detract from your specific antigen staining by masking your staining or interfering with the interpretation of your results. This is especially true if your specimen already has a lot of signal. He suggests that multiplexers avoid falling into the trap of lab dogma when following an established protocol and consider whether a counterstain is truly needed. “You can really ruin a lot of hard work [with a haphazard counterstain],” he explains. If you decide to go ahead, be very careful with the application. A light and balanced counterstain may help your specimen shine, while a heavy counterstain could muddy your results. Think carefully about the localization of your target antigen(s), too. For example, if one of your antigens is localized to the nucleus and you use a nuclear counterstain, be sure that you have a good demarcation between the counterstain and the label itself. Finally, always optimize the counterstain on separate tissue sections by troubleshooting experimental parameters such as application time and pH before applying it to your precious specimen. Ready to take the leap? Head on back to that gorgeous substrate selection page you looked at earlier to get the scoop on matching substrates to compatible counterstains!  

Right place, right time: get the hang of co-localization 

Now that you’re a multiplex immunohistochemistry pro, you may be thinking about how to reliably stain co-localized antigens. Keep a few things in mind to achieve the experimental results of your dreams. If you have 100% overlap, meaning that every cell positive for one antigen is also positive for the other, interpretation can be very challenging. Craig recommends steering clear of 100% overlap to avoid this headache. Next, remember that peroxidase and alkaline phosphatase have different physical characteristics and precipitate out in different ways. Alkaline phosphatase-based substrates yield a more diffuse and transparent stain, while peroxidase-based substrates result in a dense, punctate precipitate. Reflect on which type of system will work best for your specific co-localization experiment. Lastly, don’t forget about the order of staining—as we mentioned earlier, always target your most weakly-expressed antigen with your most sensitive detection system first. 

Follow these tips and you will be writing up your results in no time. Circle back to the SpeakEasy Science Blog soon for more expert advice to sharpen your skills at the bench, inspiring scientist stories that will brighten your day, and publication highlights to help spark your next big idea. 

1. Van Herck Y, et al. 2021. Multiplexed Immunohistochemistry and Digital Pathology as the Foundation for Next-Generation Pathology in Melanoma: Methodological Comparison and Future Clinical Applications. Frontiers in Oncology.
2. Pang L, et al. 2021. Development of a Multiplex Immunohistochemistry Workflow to Investigate the Immune Microenvironment in Mouse Models of Inflammatory Bowel Disease and Colon Cancer. International Journal of Molecular Sciences.