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Mounting Media and Antifades |
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
ProLong antifade mounting medium | SlowFade antifade mounting medium | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
ProLong Glass | NEWProLong RapidSet | ProLong Diamond | ProLong Gold | ProLong Live | SlowFade Glass | SlowFade Diamond | SlowFade Gold | |||
Counterstain availability | ProLong Glass with NucBlue | No counterstain | ProLong Diamond with DAPI | ProLong Gold with DAPI | No counterstain | SlowFade with DAPI | SlowFade with DAPI | SlowFade with DAPI | ||
Sample type | Fixed cells/tissues | Live | Fixed cells/tissues | |||||||
Curing (setting) | Curing (hard setting) | N/A | Non-curing (soft setting) | |||||||
Curing time | 18–60 hours | 1 hour | 24 hours | N/A | N/A | |||||
Imaging type | Fixed cell/tissue, long-term imaging | Fixed cell/tissue, immediate and long-term imaging | Fixed cell/tissue, long-term imaging | Live-cell imaging | Fixed cell/tissue, immediate imaging | |||||
Sample thickness | Up to 150 µm sample thickness | Up to 80 µm sample thickness | Up to 10 µm sample thickness | N/A | Up to 500 µm sample thickness | Up to 15 µm sample thickness | ||||
Refractive index | 1.51 (after 24 hr curing) | 1.49 (after 1 hr curing) | 1.47 (after 24 hr curing) | 1.3 | 1.52 | 1.42 | ||||
Optimal microscope objective* | Oil-immersion | Oil-immersion | Glycerol-corrected | Glycerol-corrected Water- or air-corrected | Glycerol-corrected | |||||
Compatible fluorophores | Most dyes and fluorescent proteins | Alexa Fluor dyes | Most dyes and fluorescent proteins | Most dyes and fluorescent proteins | Alexa Fluor dyes | |||||
Format | Ready-to-use | 100x concentration liquid | Ready-to-use | |||||||
See all ProLong antifade mountants and reagents | See all SlowFade antifade reagents |
*All objective types are compatible with these 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.
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).
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.
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.
Protocols that fit your needs in imaging ranging from sample and assay preparation to staining, labeling, and data analysis strategies.
Additionally, search through our library of online microscopy protocols.
Find helpful tools below to help you plan your imaging experiments.
SpectraViewer Supports all levels of experimental complexity, use the tool to compare excitation and emission spectra of fluorophores and reagents.
Stain-iT Cell Staining Simulator Visualize staining your cell without wasting your reagents, antibodies, or time.
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
Publications
BioProbes 73 article: ProLong Live Antifade Reagent: Protection from photobleaching for live-cell imaging
BioProbes 72 article: ProLong Diamond and SlowFade Diamond Antifade Mountants for fixed cell imaging
Posters
Antifade mounting media to help improve image quality in 3D biological samples
Cell Imaging Support Center
Find technical information, tips and tricks, and troubleshooting help for your experimental problems