Mounting Media and Antifades | Thermo Fisher Scientific

Microscopic views of fluorescence fade in cultured cells after 24 and 36 s exposure

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.

On this page:

How to select the antifade for your imaging experiment
What is photobleaching?
How to use an antifade to prevent photobleaching
What is the difference between a soft- and hard-setting mountant?

Antifades and mounts ordering information
Examples
Featured publication
Resources

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? (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

	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

Learn about techniques and methods for using an antifade, mountant or clearing reagent

Curing (hard-set)

	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? (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

Non-curing (soft set)

	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

Live cell

	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

3D cell culture and thick tissue

	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

Plant cell

	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

Learn about techniques and methods for using an antifade, mountant or clearing reagent

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

Illustration of microscope slide with sample

Step 1. Place sample on slide or coverslip. Complete all staining prior or after this step.

Illustration of microscope slide with antifade applied

Step 2. Apply antifade mountant over sample. For a hard-setting mount, allow to cure overnight open to air.

Illustration of microscope slide with glycerol applied (hard-setting mountants only)

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.

View protocol and information on how to mount specimens onto slides

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

ProLong Glass Antifade Mountants

ProLong Diamond Antifade Mountants

ProLong Gold Antifade Mountants

Slowfade Antifade Mountants

CytoVista Tissue Clearing Reagent

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)

Sountoulidis A, Liontos A, Nguyen HP, Firsova AB, Fysikopoulos A, Qian X, Seeger W, Sundström E, Nilsson M, Samakovlis C. SCRINSHOT enables spatial mapping of cell states in tissue sections with single-cell resolution. PLoS Biol. 2020 Nov 20;18(11):e3000675. PMID: 33216742.