SlowFade Antifade Reagents

SlowFade antifade reagents suppress photobleaching and preserve the signals of your fluorescently labeled target molecules for analysis. SlowFade antifade reagents do not cure over time, and samples can be viewed immediately. SlowFade reagents are intended for short-term preservation (3–4 weeks), and samples may degrade over longer periods.

Key benefits of SlowFade antifade reagents include:

  • 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
Choose the antifade reagent that matches your experiment

Performance of SlowFade antifade reagents

SlowFadeGold antifade reagent performance

SlowFade Gold antifade reagent outperforms all other commercially available antifade reagents because it preserves signal across the entire visible spectrum and causes little or no quenching of the initial fluorescent signal.

SlowFade Diamond antifade reagent performance

SlowFade Diamond antifade reagent delivers all the performance of SlowFade Gold reagent with Alexa Fluor dyes and provides additional protection for traditional dyes and fluorescent proteins. SlowFade Diamond reagent is a superior antifade and mountant, offering protection across the visible spectrum. Keep SlowFade Diamond reagent refrigerated for the best performance.

SlowFade Glass antifade reagent performance

SlowFade Glass antifade reagent is the ideal mountant for specimens with imaging depths from 0-500 µm, including cultured cells, organoids/spheroids, and thin and thick tissue sections. The mountant provides excellent protection against photobleaching across the visible and near infrared spectra, and it can be used with most fluorescent dyes or fluorescent proteins.

Figure 1. When comparing SlowFade non-curing, antifade mountants, SlowFade Diamond provides 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, 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.

Figure 2. 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 SlowFade Glass (RI ~1.52) or SlowFade Diamond (RI ~1.42) or VECTASHIELD (R1 ~1.45) 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 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.

3 panel image. 0 hrs shows opaque brain with print behind it that cannot be read. 16 and 48 hrs show a transparent brain. The print behind it can clearly be read.

Figure 3. Mouse brain clearing with SlowFade Glass. Clearing of 1-mm mouse brain section by refractive index matching with SlowFade Glass at time intervals of 0, 16, and 48 hours.

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 (Cat. No. OPA1-06100) and Alexa Fluor Plus 594 Goat Anti-Rabbit (Cat. No. A-32740) overnight. Nuclei (cyan) were stained with DAPI nuclear stain (Cat. No. D1306). Stained samples were mounted with SlowFade Glass (Cat. No. S36917), SlowFade Diamond (Cat. No. S36967), or SlowFade Gold (Cat. No. S36940) 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. Deep tissue imaging with non-curing, refractive index matched SlowFade Glass. Cryo-preserved rat brain sections (100 µm thick), stained for tubulin (red) with Mouse Anti-Beta3-Tubulin (Cat. No. MA1-118) and GFAP (yellow) Rabbit Anti-GFAP (Cat. No. OPA1-06100). Targets were detected with Alexa Fluor Plus 594 Goat Anti-Mouse (Cat. No. A-11032) and Alexa Fluor Plus 647 Goat Anti-Rabbit (Cat. No. A-32733) dyes. Nuclei (cyan) were stained with DAPI (Cat. No. D1306). Slides were mounted with non-curing SlowFade Glass Antifade Mountant (Cat. No. S36917) 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 6. Alexa Fluor 647 photobleach curves with widefield (A) and confocal (B) microscopes following treatment with SlowFade Glass.

(A) 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. 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) Tubulin in HeLa cells were 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. 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.

(A)Widefield

(B)Confocal

3D-Animation of IHC of 100 µm thick Cryo-preserved rat brain section mounted in Slowfade Glass

Cryo-preserved rat brain sections (100 µm thick), stained for tubulin (red) with Mouse Anti-Beta3-Tubulin and GFAP (yellow) Rabbit Anti-GFAP.

Find the right SlowFade antifade reagent

  SlowFade Glass SlowFade Diamond SlowFade Gold
Recommended specimen thickness Up to 500 mm Up to 15 mm Up to 15 mm
Refractive index 1.52 1.42 1.42
Cell/tissue types
  • Tissue culture cells
  • Tissue sections (FFPE and cryo-sectioned)
  • Organoid/spheroids
  • Tissue culture cells
  • Tissue sections (FFPE and cryo-sectioned)
Photobleach protection Better Best Good
Recommended for Alexa Fluor fluorophores Yes Yes Yes
Recommended for classic organic dyes (e.g. FITC, TRITC, etc.) Yes Yes No
Recommended for fluorescent proteins (e.g. GFP, RFP, etc.) Yes Yes No
Performance across the spectrum

SlowFade Diamond reagent is a superior antifade and mountant, offering protection across the visible spectrum and exhibiting less initial quenching of the fluorescent signal. It provides additional protection for traditional dyes such as FITC and fluorescent proteins such as GFP. In a comparison of commercially available soft-mount antifade reagents in a standardized experiment, SlowFade mountants outperformed other products in both confocal and bright-field applications.

Comparison of degree of photobleaching protection by SlowFade antifades for various fluorescent dyes

    Resistance to photobleaching*
Fluorophore Ex/Em (nm) SlowFade Glass SlowFade Diamond SlowFade Gold
Hoechst 33342 350/461 +++ +++ +++
DAPI 345/455 +++ +++ +++
Alexa Fluor 488 495/519 ++ +++ ++
Alexa Fluor Plus 488 495/519 ++ +++ ++
GFP 488/510 ++ +++ Not Recommended
Fluorescein 494/518 ++ +++ ++
Cy3 550/570 ++ ++ ++
Alexa Fluor 546 556/575 ++ ++ +++
Tetramethylrhodamine 555/580 +++ +++ ++
Alexa Fluor 555 555/565 +++ +++ +++
Alexa Fluor Plus 555 555/565 +++ +++ ++
TagRFP 555/584 ++ +++ Not Recommended
mCherry 575/610 ++ ++ Not Recommended
Alexa Fluor 568 578/603 +++ +++ +++
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.