ProLong Antifade Mountants for Fixed Cells | Thermo Fisher Scientific

microscopic cell image showing multicolor fluorescence

ProLong antifade mountants for fixed cells suppress photobleaching and preserve the signals of your fluorescently labeled target molecules for long-term storage and analysis. ProLong reagents cure within 12–48 hours (depending on sample thickness), forming a semi-rigid gel with a refractive index ranging from 1.47–1.52. Mounted samples can be saved for months with appropriate storage.

Key benefits of ProLong antifade mountants for fixed cells include:

Inhibit photobleaching across the spectrum, even after prolonged storage
Low background across the spectrum
Hard-setting mountants
Available with or without DAPI
Ready-to-use benchtop formulations
Choice of refractive index based on your needs: 1.52 for ProLong Glass reagent and 1.47 for ProLong Diamond and ProLong Gold reagents

Choose the antifade reagent that matches your experiment

For live-cell imaging of fluorescent dyes and proteins, see ProLong Live products
For immediate analysis and short-term storage of fixed samples see SlowFade reagents
For long-term analysis of Alexa Fluor dyes in fixed samples see ProLong Gold products
For long-term analysis of all dyes and fluorescent proteins in fixed samples see ProLong Diamond products
For long-term analysis of all dyes and fluorescent proteins in fixed samples with best optical clarity see ProLong Glass products

Performance highlights of ProLong antifade reagents for fixed cells

ProLong Gold antifade mountant performance

ProLong Gold antifade mountant has become a widely used antifade reagent for our Invitrogen high-performance fluorescent dyes and stains because it preserves signal across the entire visible spectrum and causes little or no quenching of the initial signal. ProLong Gold is room-temperature stable—keep it on the bench, ready to use in your next imaging experiment.

ProLong Diamond antifade mountant performance

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

ProLong Glass antifade mountant performance

ProLong Glass antifade mountant delivers all the performance of ProLong Diamond mountant and offers the best clarity of the ProLong product series—better resolution and improved focal depth for brighter, sharper images. ProLong Glass reagent is a superior antifade reagent with the finest clarity, offering same-as-glass refractive index of 1.52, best focal depth, and low background and photobleaching protection across the visible and near infrared spectrum. Keep ProLong Glass reagent refrigerated for best performance.

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multipanel image series showing fluorescence stained cell following repeated illumination

A 60-second time-lapse showing the enhanced resistance to photobleaching afforded by ProLong antifade mountants. Fixed HeLa cells were labeled with fluorescein phalloidin and mounted in ProLong Glass reagent, ProLong Diamond reagent, ProLong Gold reagent, or 50% PBS/glycerol. Images were acquired at 12-second intervals using a 20x objective with continuous illumination from a standard 100-watt Hg-arc lamp.

two-panel graph showing photobleaching of fluorescent signal over time

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FITC photobleach curves obtained from samples illuminated on a widefield or a confocal microscope. HeLa cells were labeled with mouse anti-tubulin primary antibody, detected with fluorescein (FITC)-labeled goat anti-mouse antibody, and mounted with PBS + 50% glycerol or various commercially available antifade mountants. (A) Photobleach curves were collected by illuminating the samples for 2 minutes using a 100‑watt Hg-arc lamp, imaged using a 20x air objective, then acquired using a 12-bit monochrome camera. The data plotted is the mean florescence intensity of three fields of view over time. (B) Photobleach curves were collected using a confocal microscope with a 20x oil-immersion objective scanning regions of interest fifty times with a pixel dwell time of 1.6 microseconds. Excitation source power intensity was set such that ProLong Diamond retained 50% of initial signal intensity at the end of the final scan. 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.

ProLong antifade mountant gallery

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Find the right ProLong antifade reagent for fixed cells

	ProLong Gold Antifade Mountant	ProLong Diamond Antifade Mountant	ProLong Glass Antifade Mountant
Alexa Fluor dyes	Yes	Yes	Yes
Traditional dyes and fluorescent proteins	No	Yes	Yes
Refractive index (after curing)	1.47	1.47	1.52
Storage temperature	Room temperature	2–8°C	2–8°C
	Ordering information	Ordering information	Ordering information

Enhanced signal preservation across the spectrum

ProLong Glass and ProLong Diamond reagents are superior antifades and mountants, offering low background and protection against photobleaching across the visible and near-infrared spectrum in addition to exhibiting less initial quenching of the fluorescent signal. It provides extra protection for traditional dyes such as FITC and fluorescent proteins such as GFP. In a comparison of commercial hard-setting antifade mountants in a standardized experiment, ProLong antifade mountants outperformed other products at preserving signals and preventing photobleaching across the spectrum.

Fluorescent dye	Ex/Em (nm)	ProLong Glass	ProLong Diamond	ProLong Gold	VECTASHIELD
Fluorescent dye	Ex/Em (nm)	Resistance to photobleaching*
DAPI	345/455	+++	+++	++	++
Hoechst	350/461	+++	+++	+++	+++
GFP	488/510	++	++	Not recommended	+
BODIPY FL	505/513	Not tested	+++	+	+
Fluorescein	494/518	+++	+++	+	++
Alexa Fluor 488	495/519	+++	++	++	++
Alexa Fluor Plus 488	495/519	+++	++	+	+
Alexa Fluor 555	555/565	+++	+++	+++	+++
Alexa Fluor Plus 555	555/565	+++	+++	++	++
Cy3	550/570	++	++	++	++
Alexa Fluor 546	556/575	++	++	+++	++
Tetramethylrhodamine	555/580	++	+++	+	+
TagRFP	555/584	Not tested	+	Not recommended	+
Alexa Fluor 568	578/603	+++	+++	+	+
mCherry	575/610	+++	+++	+++	+++
Texas Red	595/615	+++	+++	+++	++
Alexa Fluor 594	590/617	+++	+++	+++	++
TO-PRO-3	642/661	+++	+++	+	+
Alexa Fluor 647	652/668	+++	+++	+++	Not recommended
Alexa Fluor Plus 647	652/668	+++	+++	+++	Not recommended
Cy5	650/670	+++	+++	+++	++
Key: +++ = above 80%; ++ = 65—80%, + = 50—65%, Not recommended = below 50%. Percentages express remaining signal intensity. * Photobleaching resistance was quantified by measuring remaining signal intensity on a Zeiss™ LSM 710 confocal microscope. HeLa or U2OS cells were stained and mounted using standard Immunocytochemistry (ICC) protocols. Five regions within three fields of view were scanned 15 times with 1.58 μs dwell time per pixel. Excitation wavelength and intensity were optimized by fluorophore. On an epi-fluorescence microscope using 100-watt Hg-arc lamp, this amount of light/photon exposure will be equal to 60–90 seconds. VECTASHIELD is a trademark of Vector Laboratories, Inc., Burlingame, CA. Hoechst is a trademark of Hoechst, GmbH. Cy is a trademark of GE Healthcare UK, Ltd.

Get the sharpest, brightest images

Offering additional benefits for the ProLong reagent line, ProLong Glass antifade mountant features a refractive index (RI) of approximately 1.52, which is similar to that of cover glass, immersion oil, and oil objective optics. This superior optical clarity means ProLong Glass reagent delivers up to 75% improvement in axial resolution and 3–4 times more imageable focal depth compared to mountants with refractive index of 1.47, producing sharp images for monolayers of cells, tissue slices, or 3D cell cultures up to 150 μm thickness/focal depth.

confocal image series of samples mounted in different antifade mountants

ProLong Glass antifade mountant improved the depth of imaging by 40 percent, allowing nuclei to be imaged 70 μm into the tissue section. FFPE pig brain sections were processed and stained with DAPI nuclear stain overnight. Stained samples were mounted with different commercial, hard-setting antifade media according to each mounting medium's recommended protocols. Tissue sections were imaged on a Zeiss™ LSM 710 confocal microscope using a Plan-Apochromat 63x/1.4 NA Oil objective, sampling at a rate of 83 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.

graph of refractive index vs time for a series of commercial mountants

ProLong Glass Antifade Mountant cures to a final RI of ~1.52. The refractive indices of various ProLong antifade mountants were measured using a Thermo Scientific ABBE-3L refractometer by applying 300 µL of mountant and spreading to cover the measuring prism surface.

graph of resolution as a function of focal depth for samples in two mountants

ProLong Glass, with RI of 1.52, maintains a higher axial resolution than mountants with RI of 1.47. Lateral and axial resolution of 170 μm microspheres in two mountants, plotted as a point spread function (PSF) of images acquired at various focal depths. (Error bars: SD with n ≥ 3).

Axial and lateral confocal images of A549 spheroid mounted with ProLong Glass Antifade Mountant.

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For Research Use Only. Not for use in diagnostic procedures.