Keep Photobleaching at Bay

For optimal imaging of any fluorescently stained sample, the choice of mountant is critical. Of primary importance is the mountant’s antifade properties. Loss of fluorescence through irreversible photobleaching can lead to a significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration. To minimize photobleaching of fluorescently stained samples, we have developed SlowFade® Gold, SlowFade®, ProLong® Gold, and ProLong® Antifade Kits and reagents, which have been shown to increase the photostability of many of our fluorophores in fixed cells, fixed tissues, and cell-free preparations.

The selection of a suitable mountant is governed not only by its ability to minimize photobleaching, but also by its optical clarity. Here we focus on the properties of our SlowFade® Gold antifade reagent, a glycerol-based mountant designed for immediate viewing of samples. SlowFade® Gold antifade reagent effectively reduces the photobleaching rate of several commonly used fluorophores, with minimal quenching of the initial fluorescence intensity.

SlowFade® Gold Reagent Shows Minimal Fluorescence Quenching

Mountant-induced quenching of the fluorescence signal in stained samples often goes undetected because samples are typically first viewed after mounting. As compared with conventional DABCO formulations or competitor products such as VECTASHIELD® mounting medium, SlowFade® Gold antifade reagent shows superior maintenance of initial signal strength—particularly of red fluorophores—when tested side by side with a panel of fluorescent antibody conjugates on glass slides (Figure 1).

Comparison of signal retention Figure 1. Comparison of signal retention after mounting using three antifade formulations. A panel of equimolar concentrations of dyes coupled to IgG was evenly arrayed using a non-contacting spot array device (Biochip Arrayer BCA1 from PerkinElmer). The arrayed spots were then overlaid with three test mountants (SlowFade® Gold antifade reagent, VECTASHIELD® mounting medium, and a DABCO formulation) and coverslipped. Fluorescence intensities were measured before and after mounting. SlowFade® Gold antifade reagent maintains initial intensity levels over the broadest range of dyes. In contrast, VECTASHIELD® mounting medium and the DABCO formulation can quench >90% of the intensity of some red-fluorescent dyes.

Advantages of the Glycerol-Based SlowFade® Gold Reagent

Optical clarity is influenced not just by how clear a mountant is, but by how well the refractive index (RI) of the mountant, sample, and glass or other substrate are matched. RI is a measure of how fast light travels through a particular medium; when light travels between two media with different RI values, such as air and water, the path that it travels is bent, distorting the image. For example, the much lower RI of air (RI = 1.0) compared to tissue or glass is one reason why air bubbles are so undesirable when imaging samples.

Table 1 compares RI values for common media encountered in microscopy. With an RI of 1.42, SlowFade® Gold antifade reagent is well matched to the RI of cells and tissues. Glycerol-based mountants such as SlowFade® Gold reagent are especially preferred for thick samples (>15 µm), in which the RI of the tissue (~1.4) dominates the light path. Moreover, samples mounted in SlowFade® Gold reagent can be viewed immediately, and do not damage lens surfaces they may contact. Because it does not cure, SlowFade® Gold antifade reagent is intended for immediate and short-term use; drying and shrinkage can be prevented by sealing coverslip edges with nail polish, fast-drying acrylic varnish, or melted paraffin.

Table 1. Refractive indices for microscopy applications.
Media
Refractive Index
Air
1.00
Water
1.33
Crown glass
1.50–1.54
Cells and tissue
1.35–1.42
SlowFade® Gold reagent (glycerol-based)
1.42
Cured ProLong® Gold reagent
1.47
Immersion oil
1.51

Selecting the Right Mountant for Your Application

SlowFade® Gold and ProLong® Gold antifade reagents contain the same antifade formulation but different mountants (Figure 2). Samples mounted with ProLong® Gold reagent are best viewed after the mountant has cured over several days, which ensures a near-perfect RI match (~1.47) to the cover glass and immersion oil at ~1.5. This property makes ProLong® Gold reagent an excellent choice when superior optical clarity is important or when long-term (several months’) archiving is desired.

The original ProLong® and SlowFade® antifade reagents are also available. The original ProLong® Antifade Kit provides two vials—one containing the curing mountant and the other the antifade formulation—that are combined prior to use. The curing mountant can be used without the ProLong® antifade solution when photobleaching is desirable, such as for photo-acceptor bleaching controls in fluorescence resonance energy transfer (FRET) imaging.

Although it quenches some dyes, the original SlowFade® Antifade Kit provides a DABCO-based formulation in a flexible format. The SlowFade® antifade reagent is provided in 50% (v/v) glycerol, as well as in a concentrated solution without glycerol for applications in which glycerol may not be compatible—for example, membrane staining with carbocyanine dyes such as DiI.

The unique physical properties of Qdot® nanocrystals make them largely incompatible with SlowFade® Gold, ProLong® Gold, and other mountants designed for use with organic dyes. Qmount® Qdot® mounting medium is a specialized mountant that preserves the fluorescence signal of Qdot® nanocrystals with little to no quenching of the signal’s initial intensity.

SlowFade® Gold and ProLong® Gold antifade mountants Selection guide
Figure 2. Selection guide for SlowFade® Gold and ProLong® Gold antifade mountants. Both SlowFade® Gold antifade reagent and ProLong® Gold antifade reagent are also available containing the blue-fluorescent nuclear counterstain DAPI. For imaging of Qdot® nanocrystals, use Qmount® Qdot® Mounting Medium.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

For Research Use Only. Not for use in diagnostic procedures.