Considerations for Fluorescence Microscopy
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.
Because quality and intensity of the fluorescent signal of the sample must be preserved over time, alternate, additional, steps may be taken prior to- or during mounting to slow the fade of fluorescence without deterioration. Regardless, various mounting media will tend to fade with age, so diagnosing these difficulties becomes paramount to provide longevity to slides, and in maintaining stable and accurate resolution. For fluorescent mounting, several types of mounting media
and ready-to-use anti-fading kits
are commercially available that function to slow the occurrence of photobleaching. Photobleaching, in itself, is a phenomenon where exposure to the excitation light of some fluorescent labels results in diminished staining.
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
Aqueous, water-based mounting media is used when particular stains may become decolorized, or even ruined, by alcohol and xylene (e.g., for fat stains). Some variants of this category of media may even solidify, such as glycerol jelly. Recipes for glycerol jelly can be fine-tuned for specific mounting applications, though handling the medium is particularly tricky. Glycerol jelly must first be gently warmed into a liquefied state. Finally, the specimen is submerged and the cover glass is carefully placed on top.
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
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).
In some circumstances, solvent-based
mounting media must be used. Some solvent based media will solidify, and one of historical significance is Euparal (RI = 1.48). Euparal is a semi-synthetic media invented in 1904 that contains sandarac (resin from a coniferous tree, tetraclini articulate), eucalyptol, paraldehyde, camsal (camphor, and phenyl salicylate). Historically, Euparal was used to mount histological specimens and insects, and has the added advantage that specimens can be transferred directly from alcohol (a common storage medium) without the need for other solvents.
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.
Sealing coverslips is a necessary step after mounting
that close, lock in, and cure the entire slide. Sealants prevent the specimens from becoming exposed to environmental conditions, namely oxidation and degradation. A common practice for sealants utilizes a simple, inexpensive product that can be found in nearly every drug store, i.e., clear nail polish. Though nail polish may seem like an easy option as a sealant, it requires at least 30 minutes for drying time and there are numerous accounts of quenching effects, possibly linked to the water-soluble isopropyl alcohol found in some nail polishes.
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.