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Mounting Media for Microscopy
HeLa cells were stained with mouse anti-tubulin and then followed with iFluor® 610 goat anti-mouse IgG (H+L). Cells were mounted in FluoroQuest™ Mounting Medium with DAPI.
Mounting media is generally composed of three main ingredients: a base, an antifade reagent, and a plasticizer to set. The purpose of the mounting media is to protect the specimen. The mounting medium bonds the specimen, slide and coverslip together with a clear durable film to ensure strong fixation that allows for easy visibility upon sample analysis. Ideally, mounting media should be as transparent as possible and have a refractive index (RI) close to that of glass (1.51-1.54). A mounting medium with an RI too far outside this range will produce poor clarity and contrast on the slide.
Mounting Media Considerations
| Properties of an Ideal Mounting Media | |
|---|---|
| The mounting media should: | The mounting media should not: |
| Have an RI as close as possible to that of glass, and be colorless and transparent. | Cause the stain to diffuse or fade. |
| Be able to completely permeate and fill tissue interstices. | Become sticky or harden slowly. |
| Be resistant to contamination, microbial, and fungal growth. | Shrink inside the edge of the coverslip and slide upon hardening. |
| Protect from physical damage and chemical activity such as oxidation or pH changes. | Not react with, leach or induce fading in stains and reaction products. |
| Be completely miscible and combine homogeneously with the dehydrant or clearing agent. | Crystallize, crack, shrink or otherwise deform the mounted specimen. |
| Remain stable with time. | Have adverse effects on tissue components. |
Considerations for Fluorescence Microscopy
Fluorescence image of HeLa cells fixed with 4% formaldehyde then stained with Cell Navigator® F-Actin Labeling Kit. A) Image of Hela cells without adding mounting solution B) Image of Hela cells after adding ReadiUse™ microscope mounting solution. C) Image of Hela cells after adding mounting solution for 24 hours.
For immunochemical staining (including immunofluorescence and enzymatic labeling) the mounting media should be chosen by the label or chromogen used for antigen visualization. Aqueous mounting media is generally suitable for all enzymatic labels and/or chromogen combinations with fluorescent labels. Specimens mounted in this way can be mounted straight from the aqueous phase, meaning that a dehydration or clearing step is not necessary. Fluorescent emission from labeled antibodies may be influenced by certain factors of the mounting medium, including pH, ionic strength, viscosity, or the presence of quenching agents.
Antifade properties were measured using cells stained with fluorescently labeled secondary antibodies, as listed in the graph. The antifade capability of the VectaShield and FluoroQuest™ PLUS Antifade mounting mediums was measured using a 20X objective with real-time imaging over 10 sec of continuous exposure to the excitation illumination. The intensity after 10 seconds of exposure is expressed as 100% percent of the intensity at zero time. The data shown above in the graph represents the % fluorescence remaining after 3 minutes of continuous exposure.
Most antifade reagents are reactive oxygen species scavengers, and it has been shown that sometimes the antifade agent can quench the fluorescence of the dye but prolong its life at the desired intensity. p-Phenylenediamine (PPD) is a common, effective, antifade compound that may react with cyanine dyes (especially Cy2) and may potentially cleave the cyanine molecule after use. It has been noted that use of PPD may result in weak and diffusive fluorescence after prolonged storage.
Another common antifade compound, n-Propyl gallate (NPG), is non-toxic and can be used with live cells. NPG has been shown to protect against apoptosis, which must be taken into consideration if monitoring apoptosis is experimentally desired. NPG, however, is difficult to dissolve and requires prolonged heating over several hours.
A third common antifade agent includes 1,4-Diazabicyclo-octane (DABCO), also known as triethylenediamine. Though DABCO is not quite as effective as PPD, it is much less toxic and can be used in live cells. It is, however, predicted to have the same anti-apoptotic properties as NPG.
There exist a number of common mediums for fluorescent applications, including:
- Apathy's medium (RI = 1.52). Apathy's medium is widely used in microscopy as it is virtually non-fluorescent. Apathy's medium is also simple to make as the protocol is readily available.
- Farrant's medium (RI = 1.43) which has been recommended for use in fat stains.
- Highman's medium (RI = 1.52) may also be preferred and is commonly used with the metachromatic dyes (i.e., methyl violet).
Note: Additionally, Polyvinyl alcohol (PVA) may be used, common for immunofluorescence microscopy. PVA is a good alternative for glycerine jelly, and adding paraphenylenediamine to the medium has shown effectiveness in slowing photobleaching.
Water-Based Mounting Media
Fluorescence IHC of formaldehyde-fixed, paraffin-embedded Human lung adenocarcinoma positive tissue. Human lung adenocarcinoma positive tissue sections were stained with rabbit anti-EpCam antibody and then incubated with polyHRP-labeled Goat anti-Rabbit IgG secondary antibody followed by iFluor® 488 Styramide™ stain respectively. The tissue was mounted in FluoroQuest™ Anti-fading Mounting Medium with DAPI.
Note: Bubble formation is a common occurrence, and must be avoided as much as possible.
Slides prepared this way must be sealed at the edges to prevent drying. Though glycerol jelly is a more difficult medium to use, it is particularly useful for specific applications as this technique avoids the need for alcohol dehydration or other toxic organic solvents that may deform the specimen. Glycerol jelly has most notably been used for making permanent mounts of water organisms, algae, protozoa, and even pollen samples. Unlike solvent based media, glycerol jelly will not dissolve pigments such as chlorophyll, and is inexpensive, safe to handle, and quick to use with little preparation.
Table 1. FluoroQuest™ Mounting Media
| Cat# ▲ ▼ | Product Name ▲ ▼ | Unit Size ▲ ▼ |
| 20004 | FluoroQuest™ Mounting Medium with DAPI | 50 mL |
| 20005 | FluoroQuest™ Anti-fading Mounting Medium with DAPI | 20 mL |
| 44890 | FluoroQuest™ TSA/PSA Antifade Mounting Medium | 5 mL |
Other water-based mounting media will remain aqueous upon application. Though water and/or PBS have a low RI (1.333), they both provide convenient temporary mountants for some whole specimens. Additionally, glycerol may be used as a more permanent mountant. To do so, the specimen can be embedded in either a pure liquid glycerol or a glycerol media mixed with water or PBS. The addition of water and/or PBS is helpful to adjust and the RI or viscosity of the sample, if desired. To make a liquid permanent slide from glycerol, a few small drops of the media should be added into the middle of the coverslip. It is necessary to ensure there is a buffer zone between the sides of the coverslip and the media before sealing the coverslip. Sealing edges of the coverslip must be performed two to three times, and it should be noted that high concentrations of glycerol (over water and/or PBS) should be maintained to avoid the risk of contamination.
Solvent-Based Mounting Media
Schematic of idealized imaging system and focal shift. When the sample and the objective have different refractive indices, moving the sample closer to the objective does not move the effective focal plane the same distance. Brown dashed line indicates the z' axis and z' = 0 at the surface of the objective. In this schematic, F and ΔZ have opposite signs, corresponding to a situation of n2Simple Experimental Methods for Determining the Apparent Focal Shift in a Microscope System. PLoS ONE 10(8). (2015).
Euparal is safe, has quick drying times, and is noncarcinogenic. Specimens that contain water should however avoid this technique, as the mounting media may become cloudy. Another disadvantage of Euparal is that fading may occur in hematoxylin stained sections.
Another traditional solvent-based mounting media is Canada Balsam (RI= 1.52 - 1.54), that is produced naturally from the balsam fir tree, abies balsamea. Canada Balsam is a strong autofluorescent, and slides prepared with this method deteriorate very little with time. There even exist some permanent slides mounted with Canada Balsam that are over a hundred years old, and are still intact with great visibility. Canada Balsam, however, comes with an array of disadvantages. It is scarce, very expensive, slow to harden, and may become increasingly acidic over time. Upon preparation, the specimen must first be dehydrated in alcohol then placed into xylene, a toxic chemical, prior to embedding. Dammar balsam is a variant of this mounting media that has similar properties to Canada balsam, and is nowadays rarely used.
Resin-based media, namely Eukkitt, may offer an alternative solvent-based mounting media. Resins are synthetic, fast drying, ang good for general-purpose mounting. They solidify in approximately 20 minutes, can easily be constructed in a lab with an appropriate protocol, and are commercially available. To use resin-based media, first specimens must be free of water and placed in alcohol and xylene prior to mounting. Resin-based media can also be diluted with xylene to adjust the viscosity of the media where necessary, though it is important to note that some resins will shrink significantly during the drying process.
Many other resin-based mounting media are commercially available, including but not limited to Diatex, Entellan, Malinol, Rhenohistol and Depex (that differ in their RI), Histamount (RI = 1.49-1.50), Permount (RI = 1.526), Gurr's neutral mounting medium (RI = 1.51). In some cases, sections of tissue can be embedded in plastic compounds (i.e, epoxy resins) and then mounted in liquid resin of the same type. Other types of resin-based media are photosensitive and will polymerize light. One advantage of photosensitive resins is that they have extremely short setting times, requiring as little as a 10-30 second exposure to UV light to cure. These resins are permanent; after curing the mountant cannot be dissolved and the coverslip cannot be removed, which should be considered if sample restaining is desired. Other acrylic-based light sensitive resins are available and well suited for fluorescent microscopy.
Considerations for Sealants
Demonstration of microscope slide being sealed with vaseline.
Another inexpensive, readily available, option for sealants includes dental or modeling wax. These waxes are easy to apply, safe to use, and removable. Alternatively, equal mixtures of vaseline, lanolin and paraffin may be heated and mixed to form a sealant termed VALAP. VALAP is commonly used to seal living samples due to its low melting point that prevents damage to samples when applied. Commercially available sealants also exist, for example Entellan. Entellan cures solid rapidly, and is often used for sealing glycerol-based mounting media. When diluted and hard-set, Entellan can also be a useful sealant for toluene or xylene based mounting media.
Product Ordering Information
Table 2. Anti-fading kits and Mounting Mediums
| Cat# ▲ ▼ | Product Name ▲ ▼ | Unit Size ▲ ▼ |
| 20001 | FluoroQuest™ Anti-fading Kit I *Optimized for Slide Imaging* | 1 kit |
| 20003 | FluoroQuest™ Anti-fading Kit II *Optimized for Plate Imaging* | 1 kit |
| 20004 | FluoroQuest™ Mounting Medium with DAPI | 50 mL |
| 20005 | FluoroQuest™ Anti-fading Mounting Medium with DAPI | 20 mL |
| 20006 | FluoroQuest™ Fluorescence Signal Enhancing Solution | 5 mL |
| 20007 | FluoroQuest™ Antifade Mounting Medium | 5 mL |
| 20008 | FluoroQuest™ PLUS Antifade Mounting Medium | 5 mL |
| 44890 | FluoroQuest™ TSA/PSA Antifade Mounting Medium | 5 mL |
References
Ravikumar S, Surekha R, Thavarajah R. Mounting media: An overview. J NTR Univ Health Sci [serial online] 2014 [cited 2023 May 15];3, Suppl S1:1-8. https://www.jdrntruhs.org/text.asp?2014/3/5/1/128479
Different types of mounting medium for microscopy
Mounting Media and Antifade reagents