Selection of mounting media for immunohistochemistry (IHC) or immunofluorescence (IF) applications
In tissue-based histology, mounting a sample refers to the attachment of the sample onto a glass slide in preparation for microscopic examination. The mounting medium is the solution in which the sample is immersed and the right mounting medium will protect your signal and your samples while viewing. Mounting Medium might be liquid or resinous in nature, it might be intended for short- or long-term archiving, or it might possess a variety of other qualities depending on the application for which it is being used.
Vector Laboratories manufactures the industry’s best selection of mounting media for light and epi-fluorescent microscopy applications. Our mounting media are backed by independent research and optimized for either immunohistochemistry (IHC), immunofluorescence (IF), or Live Cell Imaging applications.
Explore Mounting Media
When applying a coverslip, the correct mounting medium preserves your histochemical stains or precipitable enzyme substrates in tissue sections or cell preparations.
Maximum retention of signal intensity is crucial for visualizing and imaging fluorescently stained target antigens in cell and tissue specimens. VECTASHIELD Antifade Mounting Media inhibit fading and photobleaching of most fluorophores, dyes, fluorescent proteins, and stains.
VectaCell Trolox Antifade Reagent is an antifading additive for live cell imaging. It reduces phototoxicity and photobleaching of reagents to increase cell viability and prolong signal.
All About Mounting Media
Vector Laboratories manufactures the industry’s best selection of mounting media for light and epi-fluorescent microscopy applications. Our mounting media are backed by independent research and optimized for either immunohistochemistry (IHC) or immunofluorescence (IF) experiments. The versatility of the original VECTASHIELD format addresses the challenges of labs and core facilities that use multiple platforms and fluorescent markers. VECTASHIELD reagents are also recognized as leading media in emerging techniques such as super resolution microscopy (SRM).
What’s the Purpose of Mounting Media?
At their most basic, mounting media adhere your specimen to a glass slide and coverslip and in doing so, provide a protective benefit to your sample. Some mounting media have been developed with additional benefits including:
- To prevent cells from drying out
- To prevent signals from fading or photobleaching
- To preserve specimens for short or long-term storage
- To improve the optical clarity of samples when viewed under the microscope
What are the Different Types of Mounting Media?
Mounting media can be classified as:
- Liquid mounting media, such as aqueous glycerol and lactophenol-based fluid media
- Solidifying (or semi-solid) media, including natural and synthetic gums and resins
- Water-soluble mounting media (e.g., glycerol-gelatin and gum-chloral)
- Partially water-tolerant mounting media (e.g., Euparal)
- Hydrocarbon-soluble mounting media (e.g., neutral Canada balsam)
Which Mounting Medium Should I Choose?
Choosing the ideal mounting medium for your application will require that you identify what is most important to you. Areas of consideration might be the length of time for which you intend to protect and store your sample or perhaps, whether any chemical incompatibilities exist between your sample type and the solvents of the mounting medium. If your only concern is purely to provide safe, long-term storage, you might consider a hardsetting (solidifying) medium like Canada balsam; it’s well-known for its excellent durability. The VECTASHIELD Vibrance or VectaMount products are an excellent choice for this as well.
Considerations when choosing Mounting Medium should include:
- Immunohistochemistry (IHC) or light microscopy applications: Use a colorless mounting medium, like our clear-dry VectaMount™ Permanent Mounting Medium.
- Immunofluorescence (IF) applications: Use a medium that protects the fluorescent signal with antifade, anti-photobleaching properties, like our VECTASHIELD PLUS Antifade Mounting Media
- Florijn et al. 1995. Analysis of antifading reagents for fluorescence microscopy. Cytometry. doi: 10.1002/cyto.990190213
- Jahr et al. 2016. eduSPIM: Light Sheet Microscopy in the Museum. PLOS ONE. doi: 10.1371/journal.pone.0161402
- For maximum clarity, detail, and contrast, the refractive index (RI) of the mounting medium should be close to that of glass (1.5) or the sample’s fixed/dried protein layers (1.53).
- VectaMount has an RI of 1.49 when dry and VECTASHIELD mounting media has an RI of ~ 1.45 - both superior to other commonly used reagents (e.g. Fluoromout-G has a refractive index of ~ 1.40).
To permanently mount a specimen, the use of a hardsetting medium is recommended.
- For IHC applications, we recommend VectaMount Permanent Mounting Medium. It has an RI of 1.49 when dry and is ideal for IHC applications.
- For IF applications, we recommend VECTASHIELD Vibrance. It has an ideal RI (~ 1.45), is easy to use, and eliminates the need for the sealing of coverslips.
Protection from oxidation, changes in pH, and other physical or chemical damage
Mounting media should resist microbial contamination, bubble formation, crystallization, shrinking, cracking, and deformation.
- Samples mounted with VectaMount media can be safely stored at room temperature for at least two years.
- VECTASHIELD Vibrance Medium shows no loss of signal intensity or sample integrity after weeks of storage at room temperature.
- VECTASHIELD Vibrance Mounting Mediumreduces bubble formation and retraction vs. competitor.
Some media include solvents or other chemicals that can be toxic or harm some cells, tissues, and stains. Our product-specific data sheets contain additional information about our mounting media, which can help you determine which are right for you and your specimens.
Can I Make My Own Mounting Media?
You can make your own mounting media, including glycerol-based mounting media, but creating your own mounting media can be quite challenging. Among other issues, variable and inappropriate refractive indices can make it difficult for you to know what you’re looking at under the microscope.
Independent research funded by the UK Medical Research Council noted that: “To simplify sample preparation, we have adopted protocols to mount samples for dSTORM in Vectashield , which avoids the preparation of complex chemical buffers and the need to use relatively expensive sample chambers in order to contain the liquid buffers.”
Other independent studies have commented as follows: “The mounting medium Vectashield gives thus a higher level of freedom for the selection of labelling strategy, which is important to address various biological problems.”
It might simply be easier, faster, and even less expensive to rely on our accessible, professionally made, and quality-controlled mounting media instead.
Producing immunofluorescence (IF) staining results with high specificity and sensitivity begins with choosing the best reagents. This guide will help you:
- Learn about the IF workflow and how to optimize each step
- Quickly identify and choose the most appropriate fluorescent secondary conjugates
- Select the best anti-fade medium to preserve your fluorescence signal for imaging and archiving