Superior protection from photobleaching for fixed cells

(See a list of the products featured in this article.)

The choice of mounting medium is an integral part of the fluorescence imaging workflow and is critically important when trying to obtain the highest-quality images from fixed cell and tissue samples. In immunocytochemical (ICC) and immunohistochemical (IHC) analyses, aqueous mounting media allow rapid mounting of labeled samples from water or buffer. Aqueous mounting media are generally categorized as soft- or hard-setting, depending on whether the mountant contains a gelling agent. In this article, we describe our newest antifade mounting media: hard-setting ProLong™ Diamond Mountant and soft-setting SlowFade™ Diamond Mountant. These two aqueous mounting formulations enable outstanding image quality as well as unparalleled fluorescent signal retention across the spectrum of traditional dyes, premium dyes, and even fluorescent proteins (Figure 1).

 
Figure 1. ProLong Diamond Antifade Mountant enables the acquisition of high-quality images from fixed tissues. Formalin-fixed, paraffin-embedded (FFPE) rat intestinal tissue was labeled with an antibody against histone 2B. This antibody was then detected with tyramide signal amplification using the TSA Kit #15 with Alexa Fluor™ 594 tyramide (pink). Nuclei were counterstained with NucBlue™ Fixed Cell ReadyProbes™ Reagent (blue) and then mounted in ProLong™ Diamond Antifade Mountant. This image was taken on a fluorescence microscope using a 20x objective.  

ProLong Diamond Antifade: A hard-setting mountant

ProLong Diamond Antifade Mountant is a premium hard-setting mounting medium offered in convenient 2 mL dropper bottles or in a 10 mL bottle. The mountant contains a proprietary polymeric constituent that semipermanently affixes the coverslip to the slide, allowing extended storage of the sample without the need for sealing. In addition to archivability, the polymer increases the refractive index of the mounting medium to 1.47 upon curing, close to that of glass and common immersion oils, providing greater resolution when imaging at higher magnification. The higher viscosity of the hard-setting ProLong Diamond mountant may also slow the off-rate and diffusion rate of low-affinity labels.

SlowFade Diamond Antifade: A soft-setting mountant

SlowFade Diamond Antifade Mountant is a premium soft-setting mounting medium. Unlike ProLong mountants, the SlowFade mountant contains no polymer and is instead a buffered glycerol solution. SlowFade Diamond Antifade Mountant is recommended when images must be obtained quickly, as samples can typically be imaged immediately after mounting. Also, SlowFade mountants can be washed easily from the coverslip, allowing additional staining of samples (Figure 2). Because the mountant is not permanent, longer-term storage requires sealing the coverslip with paraffin, VALAP (a mixture of Vaseline™ petroleum jelly, lanolin, and paraffin), or a solvent-based polymer mixture (such as nail polish). SlowFade Diamond mountant has a lower refractive index (1.42) than ProLong Diamond mountant; therefore, water- or glycerol-matched objectives are recommended for higher-magnification imaging.

 
Figure 2. SlowFade Diamond Antifade Mountant can be washed off easily to accommodate restaining. HeLa cells were fixed, permeabilized, blocked, probed with antibody to complex V inhibitor protein followed by Alexa Fluor™ 488 goat anti–mouse IgG (H+L) antibody, and stained with NucBlue™ Live ReadyProbes™ Reagent. Labeled cells were mounted in SlowFade™ Diamond Antifade Mountant and imaged (day 1). Slides were stored overnight at 4°C. On day 2, coverslips were removed, and cells were washed, further stained with Texas Red™ Phalloidin, remounted using SlowFade Diamond Antifade Mountant, and imaged (day 2), with no loss of signal.

Antifades that protect fluorescent dyes and proteins

ProLong Diamond and SlowFade Diamond Antifade Mountants offer superior protection from photobleaching for Alexa Fluor™ and DyLight™ dyes and traditional dyes (such as FITC, Texas Red™, and CyDye® fluorophores), as well as fluorescent proteins including emGFP, TagRFP, and mCherry (Figure 3, Figure 4). Unlike other antifade solutions, ProLong and SlowFade mountants do not significantly quench CyDye fluorophores or significantly increase background (Figure 5), helping to ensure that the best-quality high-resolution images can be obtained with the highest signal-to-noise ratios. It is important to note that fixed-cell mounting media are not intended for live-cell imaging, as the components of most fixed-cell mounting media will compromise the health of live cells or even lead to cell death. To protect live cells from photobleaching, we recommend using ProLong Live Antifade Reagent.

 
Figure 3. A 60-second time course shows the resistance to photobleaching afforded by ProLong Diamond Antifade Mountant. Fixed HeLa cells were labeled with FITC Phalloidin and mounted in ProLong™ Diamond Antifade Mountant or 50% PBS/ glycerol. Images were acquired at 12 sec intervals using a 20x objective with continuous illumination from a standard 100-watt Hg-arc lamp.  
 
Figure 4. ProLong Diamond Antifade Mountant provides unparalleled protection against photobleaching for fluorescent proteins and dyes. HeLa or U2OS cells were stained and mounted using standard ICC protocols, and photobleaching resistance was quantified on an Zeiss™ LSM 710 confocal microscope. Five regions within three fields of view were scanned 50 times with a 1.58 μsec dwell time per pixel. Excitation wavelength and intensity were optimized for the fluorophore being measured.  
  Figure 5. ProLong Diamond and SlowFade Diamond Antifade Mountants provide images with high signal-to-noise ratios (S:N). HeLa cells were fixed, permeabilized, blocked, probed with mouse anti–beta-tubulin antibody, and then labeled with Alexa Fluor™ 647 goat anti–mouse IgG (H+L) antibody. Labeled cells were mounted in the four antifade mountants indicated, stored at room temperature overnight, and then imaged using a 20x objective.