Overview of antifade mounting medium

Mounting media are essential tools in microscopy, serving to preserve and protect samples while enhancing image clarity and longevity. Mounting media can be soft- (non-curing) or hard-setting (curing), 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 re-stained 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.

There are different types of mounting media, each tailored to specific applications and imaging techniques: aqueous mounting media, non-aqueous (organic) mounting media, and antifade mounting media. Antifade mounting media is excellent for preventing photobleaching and stabilize fluorescent signals. 

Thermo Fisher Scientific has developed a series of antifade reagents that minimize photobleaching and increase fluorophore photostability in both live-cell and fixed-cell samples. We offer antifade mounting medium that allows for immediate imaging or 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. Explore our range of mounting media and reagents to find the right solution for your research and imaging requirements.

Learn more about how mounting medium can improve your images


Select the optimal antifade mounting medium for your imaging experiment

 ProLong antifade mounting mediumSlowFade antifade mounting medium
 ProLong GlassNEWProLong RapidSetProLong DiamondProLong GoldProLong LiveSlowFade GlassSlowFade DiamondSlowFade Gold
Counterstain availabilityProLong Glass with NucBlueNo counterstainProLong Diamond with DAPIProLong Gold with DAPINo counterstainSlowFade with DAPISlowFade with DAPISlowFade with DAPI
Sample typeFixed cells/tissuesLiveFixed cells/tissues
Curing (setting)Curing (hard setting)N/ANon-curing (soft setting)
Curing time18–60 hours1 hour24 hoursN/AN/A
Imaging typeFixed cell/tissue, long-term imagingFixed cell/tissue, immediate and long-term imagingFixed cell/tissue, long-term imagingLive-cell imagingFixed cell/tissue, immediate imaging
Sample thicknessUp to 150 µm sample thicknessUp to 80 µm sample thicknessUp to 10 µm sample thicknessN/AUp to 500 µm sample thicknessUp to 15 µm sample thickness
Refractive index1.51 (after 24 hr curing)1.49 (after 1 hr curing)1.47 (after 24 hr curing)1.31.521.42
Optimal microscope objective*Oil-immersionOil-immersionGlycerol-corrected

Glycerol-corrected

Water- or air-corrected

Glycerol-corrected
Compatible fluorophoresMost dyes and fluorescent proteinsAlexa Fluor dyesMost dyes and fluorescent proteinsMost dyes and fluorescent proteinsAlexa Fluor dyes
FormatReady-to-use100x concentration liquidReady-to-use
 See all ProLong antifade mountants and reagentsSee all SlowFade antifade reagents

*All objective types are compatible with these reagents


Antifade mounting medium

fluorescent cells mounted with prolong antifade

ProLong antifade mountants and reagents

ProLong antifade mountants and reagents suppress photobleaching and preserve the signals of your fluorescently labeled target molecules for long-term imaging, analysis, and archival storage.

Learn more about ProLong antifade mountants and reagents

fluorescent cells mounted with SlowFade antifade medium

SlowFade antifade reagents

SlowFade antifade reagents suppress photobleaching and preserve signals of your fluorescently labeled target molecules for immediate viewing and intended for short-term preservation (3–4 weeks).

Learn more about SlowFade antifade reagents


What is photobleaching?

Photobleaching is an irreversible process that results in 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. The mechanisms associated with photobleaching are also implicated in phototoxicity, which may adversely impact cell viability and data quality. Antifade reagents were introduced to allow for greater and longer signal by lessening the photobleaching effect.

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.


Imaging protocols


Resources

Cell Imaging Resource Center 
Access articles, webinars and videos, experimental design, and more for imaging microscopy and high-content analysis

Fixed Cell Imaging
Utilize established tools and protocols to capture and analyze high-resolution images, facilitating the study of cellular structures and architectures

Live Cell Imaging
Examine cells in their natural state to maintain cellular functions allowing for the study of various cellular processes 

Stylesheet for Classic Wide Template adjustments