Figure 1. When comparing SlowFade non-curing, antifade mountants, SlowFade Diamond antifade mountant exhibits the strongest resistance to photobleaching in a 60-second time-lapse study. Fixed HeLa cells were labeled with mouse anti-tubulin primary antibody, detected with Alexa Fluor 647-labeled goat anti-mouse antibody, and mounted in SlowFade Glass reagent, SlowFade Diamond reagent, SlowFade Gold reagent mounting reagents or 50% PBS/glycerol. Images were acquired on an EVOS M7000 Imaging system using a Cy5 Light Cube and 20x air objective with continuous illumination. Shown are images acquired at 12 second intervals.
SlowFade antifade mountants offer high-quality solutions for preserving fluorescence and maximizing imaging performance in microscopy applications. Designed to suppress photobleaching and maintain the integrity of fluorescently labeled samples, SlowFade antifade mountants offer excellent signal retention for detailed analysis. With the advantage of immediate sample viewing and no curing over time, researchers can seamlessly transition from staining to imaging without delay. SlowFade reagents are intended for short-term sample preservation (3–4 weeks). Explore our range of SlowFade antifade mountants to find the ideal solution that meets your experimental needs and elevates your imaging capabilities.
Product highlights
SlowFade antifade mountants
- Inhibit photobleaching across the spectrum
- Non-setting mountants
- Available with or without DAPI
- Ready-to-use bench-top formulations
- Refractive index of 1.52 for SlowFade Glass and 1.42 for SlowFade Diamond and Gold antifade mountants
Select the optimal SlowFade antifade mountants for your imaging experiment
Ready-to-use, soft-setting reagents used for the immediate imaging and short-term storage of fixed cells and tissues
| SlowFade Glass | SlowFade Diamond | SlowFade Gold | |||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| SlowFade Glass Soft-Set antifade mountant | SlowFade Glass Soft-Set antifade mountant with DAPI | SlowFade Diamond antifade mountant | SlowFade Diamond antifade mountant with DAPI | SlowFade Gold antifade mountant | SlowFade Gold antifade mountant with DAPI | ||||||||||||||||||||||||||||||||||||||||||||||||
| Sample type |
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| Sample thickness | Up to 500 µm sample thickness | Up to 15 µm sample thickness | |||||||||||||||||||||||||||||||||||||||||||||||||||
| Refractive index | 1.52 | 1.42 | |||||||||||||||||||||||||||||||||||||||||||||||||||
| Optimal microscope objective* | Glycerol-corrected | Glycerol-corrected | |||||||||||||||||||||||||||||||||||||||||||||||||||
| Compatible fluorophores | Most dyes and fluorescent proteins | Most dyes and fluorescent proteins | Alexa Fluor dyes | ||||||||||||||||||||||||||||||||||||||||||||||||||
| Reagent storage temperature | –20°C | 2–8°C | Room temperature | ||||||||||||||||||||||||||||||||||||||||||||||||||
| Sizes | 5 x 2 mL 2 mL | 5 x 2 mL 2 mL 10 mL | 5 x 2 mL 2 mL 10 mL | ||||||||||||||||||||||||||||||||||||||||||||||||||
| User guide | User Guide: SlowFade Antifade Mountants (non-curing, soft set) | ||||||||||||||||||||||||||||||||||||||||||||||||||||
*All objective types are compatible with these reagents
Ordering information
Performance of SlowFade antifade mountants
Suppress photobleaching to preserve fluorescent signal
During fluorescence microscopy, the prolonged exposure to light can lead to photobleaching. This can result in decreased signal intensity and negatively affect the quality of images and reliability of data. SlowFade antifade reagents suppress photobleaching and preserve the signals of your fluorescently labeled samples. SlowFade Diamond antifade mountant provides the best photostability resistance among the SlowFade antifade mountant product series (Figure 1, 2).
Figure 2. Alexa Fluor 647 photobleach curves with widefield (A) and confocal (B) microscopes following treatment with SlowFade Glass antifade mountant. Tubulin in HeLa cells was labeled with mouse anti-tubulin primary antibody, detected with Alexa Fluor 647-labeled goat anti-mouse antibody, and mounted with PBS + 50% glycerol or various SlowFade non-curing antifade mounting media. (A) Photobleach curves were collected by illuminating the samples for 1 minute using an EVOS M7000 Imaging system using a Cy5 Light Cube and 20x air objective with continuous illumination. (B) Photobleach curves were collected using a confocal microscope with a 20x air objective scanning regions of interest fifty times with a pixel dwell time of 1.6 µs. The 633 nm excitation source power intensity was set to maximum. Detector gain was held constant for all mounting media. Plotted data is the mean fluorescence intensity from fifteen regions of interest across mounted samples as number of scans.
Higher refractive index for increased focal depth
The refractive index (RI) is a crucial property in imaging applications as it quantifies a material's ability to bend or refract light. If there are significant differences in RI between the specimen and the mounting medium, it can cause optical distortions, or spherical aberrations. Controlling and understanding how light interacts with various materials is vital for capturing clear and precise images. SlowFade Glass soft-set antifade mountant enables the best refractive index of the SlowFade product series, improving axial resolution, focal depth, and image quality. With a RI of 1.52, the suppression of photobleaching minimalizes spherical aberrations, leading to improved axial resolution by up to three times at a focal point of 150 µm when compared to mountants with lower refractive indices (Figure 3). Additionally, the higher RI and axial resolution enhances clarity and sharpness at different focal depths, allowing the ability to capture and distinguish fine details at varying depths within the sample (Figure 4, 5, video).
Figure 3. SlowFade Glass mountant retains the best axial/XZ resolution at 150 micron focal depth compared to mountants of lower refractive index. To detect the lateral and axial resolution at shallow and deep focal depths, sub-resolution fluorescent yellow (Ex/Em 505 nm/515 nm) 170-nm microspheres were absorbed onto the surface of a glass coverslip and a microscope slide. Two pieces of tape were stacked and used as spacers to position mounted coverslips (Zeiss™ high tolerance #1.5 170 nm ± 5 nm) approximately 150 µm from the microscope slide. Microspheres were mounted in SlowFadeGlass (RI ~1.52) or SlowFade Diamond (RI ~1.42) or VECTASHIELD (R1 ~1.45) mountant and coverslips were adhered to the microscope slides with paraffin. Plotted data shows axial and lateral resolutions as a function of focal depth for microspheres absorbed to the coverslip (0 µm) and microscope slide (150 µm). SlowFade Glass mountant with a refractive index of ~1.52 maintains a higher axial resolution than mountants of 1.42 and 1.45 refractive index at 150 µm focal dept. Lateral resolution remains the same in all mountants at all focal depths tested as expected. The maximum theoretical axial resolution of the microscope is 500 nm, with 200 nm for lateral direction.
Figure 4. Improved focal depth in 100 µm-thick brain tissue sections with SlowFade Glass Antifade Mounting Media. Cryo-preserved 100 µm-thick rat brain sections were stained for GFAP (red) with Rabbit Anti-GFAP and Alexa Fluor Plus 594 Goat Anti-Rabbit overnight. Nuclei (cyan) were stained with DAPI nuclear stain. Stained samples were mounted with SlowFade Glass, SlowFade Diamond, or SlowFade Gold non-curing antifade mounting media. Tissue sections were imaged on a Zeiss™ LSM 710 confocal microscope using a Plan-Apochromat 63×/1.4 NA Oil objective sampling at a rate of 71 nm in the x and y dimensions and 100 nm in the z dimension, with a pixel size of 0.07 μm. Z-projections were generated using Zeiss™ Zen software.
Figure 5. Mouse brain clearing with SlowFade Glass antifade mountant. Clearing of 1-mm mouse brain section by refractive index matching with SlowFade Glass antifade mountant time intervals of 0, 16, and 48 hours.
3D-Animation of IHC of 100 µm thick Cryo-preserved rat brain section mounted in Slowfade Glass mountant.
Cryo-preserved rat brain sections (100 µm thick), stained for tubulin (red) with Mouse Anti-Beta3-Tubulin and GFAP (yellow) Rabbit Anti-GFAP.
Performance across the spectrum
SlowFade Diamond reagent is an excellent antifade and mountant, offering protection across the visible spectrum and exhibiting less initial quenching of the fluorescent signal. It enables additional protection for traditional dyes such as FITC and fluorescent proteins such as GFP. SlowFade Glass and SlowFade Diamond reagent can be used with most fluorescent dyes and proteins, while SlowFade Gold reagent is optimal for use with Alexa Fluor dyes (Table 1).
Table 1. Comparison of degree of photobleaching protection by SlowFade antifades for various fluorescent dyes
| Fluorescent dye | Ex/Em (nm) | Resistance to photobleaching* | ||
|---|---|---|---|---|
| SlowFade Glass | SlowFade Diamond | SlowFade Gold | ||
| DAPI | 345/455 | +++ | +++ | +++ |
| Hoechst 33342 | 350/461 | +++ | +++ | +++ |
| GFP | 488/510 | ++ | +++ | Not recommended |
| Fluorescein | 494/518 | ++ | +++ | ++ |
| Alexa Fluor 488 | 495/519 | ++ | +++ | ++ |
| Alexa Fluor Plus 488 | 495/519 | ++ | +++ | ++ |
| Alexa Fluor 555 | 555/565 | +++ | +++ | +++ |
| Alexa Fluor Plus 555 | 555/565 | +++ | +++ | ++ |
| Cy3 | 550/570 | ++ | ++ | ++ |
| Alexa Fluor 546 | 556/575 | ++ | ++ | +++ |
| Tetramethylrhodamine | 555/580 | +++ | +++ | ++ |
| TagRFP | 555/584 | ++ | +++ | Not recommended |
| Alexa Fluor 568 | 578/603 | +++ | +++ | +++ |
| mCherry | 575/610 | ++ | ++ | Not recommended |
| Texas Red | 595/615 | +++ | +++ | +++ |
| Alexa Fluor 594 | 590/617 | +++ | +++ | +++ |
| Alexa Fluor Plus 594 | 590/617 | +++ | +++ | +++ |
| TO-PRO-3 | 642/661 | ++ | +++ | ++ |
| Alexa Fluor 647 | 652/668 | +++ | +++ | +++ |
| Alexa Fluor Plus 647 | 652/668 | +++ | +++ | +++ |
| Cy5 | 650/670 | +++ | +++ | +++ |
| *The degree of photobleaching protection is compiled from a combination of bright field exposure and confocal scanning data to represent typical user experience in fluorescence imaging applications. | ||||
Image gallery of SlowFade antifade mountant reagents
Figure 6. 100 µm thick-tissue samples were stained and mounted with SlowFade Glass reagent. Cryo preserved rat brain tissue section immunostained for GFAP with rabbit anti-GFAP and detected using Alexa Fluor 594 Plus goat anti-rabbit. Nuclei were counter-stained with DAPI. A 63X oil immersion objective was used and the resulting image represented as a maximum intensity projection.
Figure 7. 100 µm thick-tissue samples were stained and mounted with SlowFade Glass reagent. Mouse mammary tissue section probed with mouse anti-histone H3 and rabbit anti-pan actin. Targets were detected with Alexa Fluor 488 Goat Anti-Mouse and Alexa Fluor 594 Goat Anti-Rabbit secondary antibody conjugates. The tissue was counter-stained with SYTOX DR dye. A 40X oil immersion objective was used and the resulting image represented as a maximum intensity projection.
Figure 8. Cryo-preserved tissue section mounted with SlowFade Glass reagent. Cryo-preserved rat brain sections (100 µm thick), stained for tubulin (red) with Mouse Anti-Beta3-Tubulin and GFAP (yellow) Rabbit Anti-GFAP. Targets were detected with Alexa Fluor Plus 594 Goat Anti-Mouse and Alexa Fluor Plus 647 Goat Anti-Rabbit dyes. Nuclei (cyan) were stained with DAPI. Slides were mounted with non-curing SlowFade Glass antifade mountant and imaged with a Zeiss™ LSM 710 confocal microscope using a Plan-Apochromat 63×/1.4 NA Oil immersion objective at a rate of 71 nm in the x and y dimensions and 110 nm in the z dimension, with a pixel size of 0.07 μm. Z-projections were generated using Zeiss™ Zen software.
Figure 9. HeLa cells mounted with SlowFade Diamond mountant. HeLa cells were fixed, permeabilized and blocked, stained with ActinGreen 488 ReadyProbes Reagent, NucBlue Live ReadyProbes Reagent, and AlexaFluor 647 Goat Anti-Rabbit IgG (H+L) Antibody against peroxisomes, and mounted using SlowFade Diamond mountant.
Figure 10. Skin fibroblast mounted with SlowFade Gold mountant. The actin cytoskeleton of a fixed and permeabilized muntjac skin fibroblast was labeled with rhodamine phalloidin, the nucleus was stained with DAPI. The sample was mounted and image in SlowFade Gold antifade mountant.