VECTASHIELD® Antifade Mounting Media
Antifade mounting media optimized for immunofluorescence applications
Choosing an effective mounting medium is especially important for immunofluorescence imaging. Fluorophores are susceptible to photobleaching and fading from the imaging excitation light, handling, and during storage.
Reference table indicating comparative excitation and signal retention properties for various VECTASHIELD Antifade mounting media formulations (non-setting and setting / curing).
Venn diagram showcasing the applications for which our various VECTASHIELD antifade mounting media are recommended.
All About VECTASHIELD® Antifade 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).
Table of Contents
- What are Mounting Media?
- What's the Purpose of Mounting Media?
- What are the Different Types of Mounting Media?
- Which Mounting Media Should I Choose?
- Can I Make My Own Mounting Media?
What are Mounting Media?
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 that solution might be liquid or resinous in nature, and it may possess a variety of other qualities depending on the application for which it is being used.
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)
Identifying the appropriate mounting media for your application will depend on numerous factors such as how long 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 that of long-term storage, you might consider a hardsetting (solidifying) medium like Canada balsam; it’s well-known for its excellent durability.
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. In making this determination, topics of consideration for you 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.
VECTASHIELD Antifade Mounting Media are well-regarded for their antioxidant-like, free-radical scavenging capabilities.
This analysis of antifading reagents for fluorescence microscopy conducted with a grant from the Netherlands Organisation for Scientific Research found that, “Vectashield offered the best antifading properties [with respect to study comparators and]...Vectashield is particularly recommended as a mounting medium for quantitative digital imaging microscopy and for multicolor applications.”
Additionally, this study about Light Sheet Microscopy in the Museum mentioned that “[for] mounting in Vectashield, overall photobleaching was greatly reduced and even negligible during dark periods.”
- 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 create a permanent mount of a specimen, use a hardsetting medium. The medium should be able to harden quickly but still provide an ideal RI.
- VectaMount Permanent Mounting Medium has an RI of 1.49 when dry and is ideal for IHC applications
- VECTASHIELD viscous and hardsetting mounting media eliminate the hassle of securing and sealing coverslips and mounting media with substances like nail polish, which can distort imagery in some cases
- For IF applications, use VECTASHIELD® HardSet™ Antifade Mounting Medium, which hardens in as little as 20 minutes at room temperature!
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 Medium reduces bubble formation and retraction vs. competitor.
- Some media include solvents or other chemicals that can be toxic or harm some cells, tissues, and stains. See our product-specific data sheets for more information on which of our mounting media 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