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Prevent fluorescence photobleaching
Loss of fluorescence through irreversible photobleaching processes 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. The mechanisms associated with photobleaching are also implicated in phototoxicity, which may adversely impact cell viability and data quality.
To minimize photobleaching of experimental samples, we have developed a series of antifade reagents that increase fluorophore photostability in both live-cell and fixed-cell samples. We offer choices for immediate imaging and for long-term storage, plus formulations that include a nuclear stain to combine mounting and counterstaining in a single step. Reduced photobleaching means longer tracking for time-course experiments, higher sensitivity, and better quantitation from fluorescent signals.
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How to select the antifade for your imaging experiment
| ProLong Glass | ProLong Diamond | ProLong Gold | |
|---|---|---|---|
| Learn more | |||
| General specifications | |||
| Form | Ready-to-use hard-setting mountant | ||
| Refractive index | 1.52 (after curing) | 1.47 (after curing) | 1.47 (after curing) |
| What type of imaging am I planning? | |||
| Specimen type | Animal cells/tissue (fixed) | ||
| Live-cell imaging | |||
| Fixed-cell, immediate imaging | |||
| Fixed-cell, long-term imaging (up to 10 µm sample thickness) |  | | |
| Fixed-cell, long-term imaging (up to 150 µm sample thickness) | | ||
| What type of microscope objective can produce best results?
| |||
| Oil immersion objective | | ||
| Glycerol-corrected objective | | | |
| Water- or air-corrected objective | |||
| What fluorophores does my sample include? | |||
| Alexa Fluor dyes | | | |
| Traditional dyes | | | |
| Fluorescent proteins | | | |
| Do I want to add counterstain in the mountant? | |||
| Counterstain with DAPI | | | |
| Counterstain with SYTOX Deep Red | | | |
| No counterstain | | | |
| SlowFade Glass | SlowFade Diamond | SlowFade Gold | |
|---|---|---|---|
| Learn more | |||
| General specifications | |||
| Form | Ready-to-use soft-setting mountant | ||
| Refractive index | 1.52 | 1.42 | 1.42 |
| What type of imaging am I planning? | |||
| Specimen type | Animal cells/tissue (fixed) | ||
| Live-cell imaging | |||
| Fixed-cell, immediate imaging | | | |
| Fixed-cell, long-term imaging (up to 10 µm sample thickness) | |||
| Fixed-cell, long-term imaging (up to 500 µm sample thickness) | | ||
| What type of microscope objective can produce best results? (All objective types are compatible with these reagents) | |||
| Oil immersion objective | |||
| Glycerol-corrected objective | | | |
| Water- or air-corrected objective | |||
| What fluorophores does my sample include? | |||
| Alexa Fluor dyes | | | |
| Traditional dyes | | | |
| Fluorescent proteins | | | |
| Do I want to add counterstain in the mountant? | |||
| Counterstain with DAPI | | | |
| Counterstain with SYTOX Deep Red | |||
| No counterstain | | | |
| ProLong Live | |
|---|---|
| Learn more | |
| General specifications | |
| Form | 100x concentrated liquid |
| Refractive index | 1.3 |
| What type of imaging am I planning? | |
| Specimen type | Animal cells/tissue (live) |
| Live-cell imaging | |
| Fixed-cell, immediate imaging | |
| Fixed-cell, long-term imaging (up to 10 µm sample thickness) | |
| Fixed-cell, long-term imaging (up to 150 µm sample thickness) | |
| What type of microscope objective can produce best results? (All objective types are compatible with these reagents) | |
| Oil immersion objective | |
| Glycerol-corrected objective | |
| Water- or air-corrected objective | |
| What fluorophores does my sample include? | |
| Alexa Fluor dyes | |
| Traditional dyes | |
| Fluorescent proteins | |
| Do I want to add counterstain in the mountant? | |
| Counterstain with DAPI | |
| Counterstain with SYTOX Deep Red | |
| No counterstain | |
| ProLong Glass (curing) | SlowFade Glass (non-curing) | CytoVista Reagents (non-curing) | |
|---|---|---|---|
| Learn more | Learn more | Learn more | |
| General Specifications | |||
| Form | Ready-to-use, curing (hard-setting) mountant For immediate imaging of samples up to 150 μm thick | Ready-to-use non-curing (soft-set) mountant For immediate imaging of samples up to 500 μm thick | Ready-to-use, non-curing (soft-setting) family of reagents 3D Cell Culture clearing and staining kit for immediate imaging of 3D cell culture (organoids and spheroids up to 1 mm thick) Tissue clearing and staining kit for immediate imaging of thick tissue (up to 10 mm) |
| Refractive index | 1.52 (after curing) | 1.52 | 1.48 (organoids/spheroids up to 1 mm thick) 1.53 (tissue samples up to 10 mm thick) |
| What type of imaging am I planning? | |||
| Specimen type | Animal cells/tissue (fixed) | Animal cells/tissue (fixed) | Animal cells/tissue (fixed) |
| Fixed-cell imaging, immediate imaging | | | |
| Fixed-cell, long-term imaging | | ||
| What is the thickness of my sample? | |||
| Up to 10 μm | | ||
| Up to 150 μm | | ||
| Up to 500 μm | | ||
| Up to 1 mm | | ||
| Up to 10 mm | | ||
| What type of microscope objective can produce best results? (All objective types are compatible with these reagents) | |||
| Oil immersion objective | | | |
| Glycerol-corrected objective | | ||
| What fluorophores does my sample include? | |||
| Alexa Fluor dyes | | | |
| Traditional dyes | | | |
| Fluorescent proteins | | | |
| Do I want to add counterstain in the mountant? | |||
| Counterstain with DAPI | | | |
| Counterstain with SYTOX Deep Red | | ||
| No counterstain | | | |
| Image-iT Plant Clearing and Mounting Reagents for Imaging | |
|---|---|
| Learn more | |
| General Specifications | |
| Form | Ready-to-use reagents for clearing and mounting plant tissue for imaging |
| Refractive index | 1.45 (clearing and soft-set mounting option) 1.415 (hard-set mounting option) |
| What type of imaging am I planning? | |
| Specimen type | Plant cells/tissue section/whole tissue (fixed) |
| Live-cell imaging | |
| Fixed-cell imaging | |
| Fixed-cell imaging, long-term imaging (up to 10 μM sample thickness) | |
| Fixed-cell imaging, long-term imaging (up to 150 μM sample thickness) | |
| What type of microscope objective can produce best results? (All objective types are compatible with these reagents) | |
| Oil immersion objective | |
| Glycerol-corrected objective | |
| Water- or air-corrected objective | |
| What fluorophores does my sample include? | |
| Most common histological stains | |
| Fluorescent proteins | |
| Do I want to add counterstain in the mountant? | |
| Counterstain with DAPI | |
| Counterstain with SYTOX Deep Red | |
| No counterstain | |
| ProLong Glass | ProLong Diamond | ProLong Gold | |
|---|---|---|---|
| Learn more | |||
| General specifications | |||
| Form | Ready-to-use hard-setting mountant | ||
| Refractive index | 1.52 (after curing) | 1.47 (after curing) | 1.47 (after curing) |
| What type of imaging am I planning? | |||
| Specimen type | Animal cells/tissue (fixed) | ||
| Live-cell imaging | |||
| Fixed-cell, immediate imaging | |||
| Fixed-cell, long-term imaging (up to 10 µm sample thickness) | | | |
| Fixed-cell, long-term imaging (up to 150 µm sample thickness) | | ||
| What type of microscope objective can produce best results?
| |||
| Oil immersion objective | | ||
| Glycerol-corrected objective | | | |
| Water- or air-corrected objective | |||
| What fluorophores does my sample include? | |||
| Alexa Fluor dyes | | | |
| Traditional dyes | | | |
| Fluorescent proteins | | | |
| Do I want to add counterstain in the mountant? | |||
| Counterstain with DAPI | | | |
| Counterstain with SYTOX Deep Red | | | |
| No counterstain | | | |
| SlowFade Glass | SlowFade Diamond | SlowFade Gold | |
|---|---|---|---|
| Learn more | |||
| General specifications | |||
| Form | Ready-to-use soft-setting mountant | ||
| Refractive index | 1.52 | 1.42 | 1.42 |
| What type of imaging am I planning? | |||
| Specimen type | Animal cells/tissue (fixed) | ||
| Live-cell imaging | |||
| Fixed-cell, immediate imaging | | | |
| Fixed-cell, long-term imaging (up to 10 µm sample thickness) | |||
| Fixed-cell, long-term imaging (up to 500 µm sample thickness) | | ||
| What type of microscope objective can produce best results? (All objective types are compatible with these reagents) | |||
| Oil immersion objective | |||
| Glycerol-corrected objective | | | |
| Water- or air-corrected objective | |||
| What fluorophores does my sample include? | |||
| Alexa Fluor dyes | | | |
| Traditional dyes | | | |
| Fluorescent proteins | | | |
| Do I want to add counterstain in the mountant? | |||
| Counterstain with DAPI | | | |
| Counterstain with SYTOX Deep Red | |||
| No counterstain | | | |
| ProLong Live | |
|---|---|
| Learn more | |
| General specifications | |
| Form | 100x concentrated liquid |
| Refractive index | 1.3 |
| What type of imaging am I planning? | |
| Specimen type | Animal cells/tissue (live) |
| Live-cell imaging | |
| Fixed-cell, immediate imaging | |
| Fixed-cell, long-term imaging (up to 10 µm sample thickness) | |
| Fixed-cell, long-term imaging (up to 150 µm sample thickness) | |
| What type of microscope objective can produce best results? (All objective types are compatible with these reagents) | |
| Oil immersion objective | |
| Glycerol-corrected objective | |
| Water- or air-corrected objective | |
| What fluorophores does my sample include? | |
| Alexa Fluor dyes | |
| Traditional dyes | |
| Fluorescent proteins | |
| Do I want to add counterstain in the mountant? | |
| Counterstain with DAPI | |
| Counterstain with SYTOX Deep Red | |
| No counterstain | |
| ProLong Glass (curing) | SlowFade Glass (non-curing) | CytoVista Reagents (non-curing) | |
|---|---|---|---|
| Learn more | Learn more | Learn more | |
| General Specifications | |||
| Form | Ready-to-use, curing (hard-setting) mountant For immediate imaging of samples up to 150 μm thick | Ready-to-use non-curing (soft-set) mountant For immediate imaging of samples up to 500 μm thick | Ready-to-use, non-curing (soft-setting) family of reagents 3D Cell Culture clearing and staining kit for immediate imaging of 3D cell culture (organoids and spheroids up to 1 mm thick) Tissue clearing and staining kit for immediate imaging of thick tissue (up to 10 mm) |
| Refractive index | 1.52 (after curing) | 1.52 | 1.48 (organoids/spheroids up to 1 mm thick) 1.53 (tissue samples up to 10 mm thick) |
| What type of imaging am I planning? | |||
| Specimen type | Animal cells/tissue (fixed) | Animal cells/tissue (fixed) | Animal cells/tissue (fixed) |
| Fixed-cell imaging, immediate imaging | | | |
| Fixed-cell, long-term imaging | | ||
| What is the thickness of my sample? | |||
| Up to 10 μm | | ||
| Up to 150 μm | | ||
| Up to 500 μm | | ||
| Up to 1 mm | | ||
| Up to 10 mm | | ||
| What type of microscope objective can produce best results? (All objective types are compatible with these reagents) | |||
| Oil immersion objective | | | |
| Glycerol-corrected objective | | ||
| What fluorophores does my sample include? | |||
| Alexa Fluor dyes | | | |
| Traditional dyes | | | |
| Fluorescent proteins | | | |
| Do I want to add counterstain in the mountant? | |||
| Counterstain with DAPI | | | |
| Counterstain with SYTOX Deep Red | | ||
| No counterstain | | | |
| Image-iT Plant Clearing and Mounting Reagents for Imaging | |
|---|---|
| Learn more | |
| General Specifications | |
| Form | Ready-to-use reagents for clearing and mounting plant tissue for imaging |
| Refractive index | 1.45 (clearing and soft-set mounting option) 1.415 (hard-set mounting option) |
| What type of imaging am I planning? | |
| Specimen type | Plant cells/tissue section/whole tissue (fixed) |
| Live-cell imaging | |
| Fixed-cell imaging | |
| Fixed-cell imaging, long-term imaging (up to 10 μM sample thickness) | |
| Fixed-cell imaging, long-term imaging (up to 150 μM sample thickness) | |
| What type of microscope objective can produce best results? (All objective types are compatible with these reagents) | |
| Oil immersion objective | |
| Glycerol-corrected objective | |
| Water- or air-corrected objective | |
| What fluorophores does my sample include? | |
| Most common histological stains | |
| Fluorescent proteins | |
| Do I want to add counterstain in the mountant? | |
| Counterstain with DAPI | |
| Counterstain with SYTOX Deep Red | |
| No counterstain | |
What is photobleaching?
Photobleaching is the degradation of fluorescent signal. Free radicals are generated when photoexcited fluorophores are exposed to oxygen, leading to the loss of signal intensity. Photobleaching can be slowed by reducing the intensity and time of exposure of the fluorophore to light. Antifade reagents were introduced to allow for greater and longer signal by lessening the photobleaching effect.
How to use an antifade to prevent photobleaching
Step 1. Place sample on slide or coverslip. Complete all staining prior or after this step.
Step 2. Apply antifade mountant over sample. For a hard-setting mount, allow to cure overnight open to air.
Step 3. Only with a hard-setting mountant: add a drop of 100% glycerol to the mounted sample and apply a coverslip. Glycerol aids in coverslip adhesion. ProLong Glass antifade mountant fully cures to a final RI of ~1.52 after 24 hours.
What is the difference between a soft- and hard-setting mountant?
Mounting media can be soft- or hard-setting, depending on whether the mountant contains a gelling agent. Soft-setting mountants contain a buffered glycerol solution and should be used when slides need to be imaged immediately. Soft-setting mountants can be washed away and the sample can either be restained or used for downstream purposes like single-cell RNA-sequencing or PCR. Hard-setting mountants contain a polymer that permanently affixes the coverslip to the slide. This is used for extended storage of a sample.
Antifades and mounts ordering information
Examples of samples mounted with ProLong Antifade Mountants and Slowfade Antifade Mountants
Slowfade Antifade Mountant Example
Figure 1. 100 micron thick-tissue samples were stained and mounted with SlowFade Glass and Images were acquired a Zeiss LSM 710 confocal microscope. Left) 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. Right) 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 (Cat. No. A11029) and Alexa Fluor 594 Goat Anti-Rabbit (Cat. No. A11012) secondary antibody conjugates. The tissue was counter-stained with SYTOX DR (Cat. No. S11381). A 40X oil immersion objective was used and the resulting image represented as a maximum intensity projection.
ProLong Antifade Mountant Example
Figure 2. Cryo-preserved rat brain sections (100-µm thick) were 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). Nuclei (cyan) were stained with DAPI (Left, Cat. No. D1306). Slides were mounted with ProLong Glass Antifade Mountant and imaged with a Zeiss LSM 710 confocal microscope using 63×oil Immersion.
Featured publication
Publication showing stripping and FISH reprobing after imaging with SlowFade Gold Antifade Mountant (Cat. No. S36936)
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