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ProLong™ Glass Antifade Mountant with NucBlue™ Stain (Invitrogen™)

ProLong Glass Antifade Mountant is a glycerol-based, hard-setting, ready-to-use mountant that can be applied directly to fluorescently labeled cells or tissue samples on microscope slides or coverslips. Prolong Glass mountant has a refractive index of 1.52 after curing, similar to that of glass coverslips, compatible immersion oil, and oil-immersion microscope optics, enabling superior resolution and sensitivity. ProLong Glass mountant is also designed to provide unparalleled photobleach protection across the visible and near infra-red spectra. You can use ProLong Glass Antifade Mountant with almost any fluorescent dye or fluorescent protein (e.g., GFP, RFP, mCherry) and with any cell/tissue type for image depths ranging from 0 to 100 µm for bright, high-resolution Z-Stack, 3D, and 2D images.

With its high refractive index of 1.52 and superior antifade formulation, ProLong Glass Antifade Mountant delivers the following benefits:
• Up to 75% improvement in axial resolution for sharp images
• 3–4 times more imageable focal depth compared tomountants with refractive index of 1.47, with sharp images for monolayers of cells, tissue slices, or 3D cell cultures up to 150 μm thickness/focal depth
• Low background across the spectrum
• Preserved signals from inhibited photobleaching
• Weeks to months of stability for slide-mounted samples
• Ease of use
• Availability with or without DAPI

Learn more about ProLong Antifade mountants >

ProLong GlassAntifade Mountant comes already mixed and ready to use in a dropper bottle or 10-mL bottle with or without NucBlue (Hoechst 33342) stain, a cell-permeant nuclear counterstain that emits blue fluorescence when bound to DNA. After removing excess water, just apply a drop of mountant to the sample, add a coverslip, cure, and image. ProLong Glass mountant is a hard-setting, curing mountant that minimizes optical refraction and spherical aberration in the optical path, enables longer-term storage of your samples, and helps protect your samples from photobleaching. ProLong Glass mountant does not discolor or shrink when cured, making it possible to take high-quality fluorescent images weeks, or even months, after mounting the slides.

ProLong Glass Antifade Mountant with NucBlue stain contains an optimized concentration of Hoechst 33342 stain, which provides superior nuclear labeling of most cells and tissues. ProLong Glass mountant with NucBlue stain is not recommended for specimens thicker than 30 µm. Thicker specimens can require longer incubations with labeling reagents, so a pre-incubation with NucBlue Live ReadyProbes Reagent is recomended.

Brightest and sharpest images
Studies show that the axial resolution of a microscopy system can be improved by matching the refractive index of the mounting medium, which maximizes light collection of the objective lens (1–3). Fouquet et. al. saw dramatic improvement in axial resolution while using a confocal microscope with high refractive index media (3). When Prolong Glass Antifade Mountant, which has a refractive index of 1.52, is compared with a mounting medium with a refractive index of 1.47, the axial resolution is improved by 32% at a focal depth of 40 µm and 75% at a focal depth of 100 µm (see figure below). The signal intensity and sensitivity of fluorophores is also improved as the focal depth increases within the sample. The same or greater improvement in resolution and sensitivity is seen in the lateral direction. This makes Prolong Glass a superior mountant for acquisition of Z-Stack, 3D, and 2D images of cells and tissue at depths from 0 to 100 µm (see figure below).

Inhibit photobleaching across the visible and near-IR spectra
No matter which fluorescent dyes or fluorescent proteins you are using, ProLong Glass Antifade Mountant helps provide superior protection against photobleaching. It has been shown to have best-in-class performance with a wide array of fluorescent dyes, including Alexa Fluor, FITC, TRTC, Texas Red, Cy3, and Cy5 dyes (see figure below). ProLong Glass mountant does not quench fluorophores, and its ability to provide brighter signal against no background helps ensure that you get the best-quality high-resolution images for publishing.

Stable signals during imaging and for longer-term storage
ProLong Glass Antifade Mountant is the ideal option for archiving samples with fluorescent dyes or fluorescent proteins and for preserving signal strength during prolonged imaging sessions, such as repeated scanning under confocal laser scanning illumination or with super-resolution microscopy. And it's easy to use—simply remove excess water from the stained sample and mount. For immediate viewing of the sample, choose our non-curing mountant: SlowFade Diamond Antifade Mountant.

Achieve excellent refractive index matching for crisp images
For modern high-resolution imaging applications, refractive index matching is critical. ProLong Glass Antifade Mountant cures to a refractive index of 1.52. Additionally, this curing property eliminates the need to use messy acrylic resins (i.e., fingernail polish), which can damage or ruin modern microscope objectives.

References
1: Cremer C, Masters BR. Resolution enhancement techniques in microscopy. Eur Phys J. 2013 38(3):281-344.
2: Shimi T, Kittisopikul M, Tran J, Goldman AE, Adam SA, Zheng Y, Jaqaman K, Goldman RD. Structural organization of nuclear lamins A, C, B1, and B2 revealed by superresolution microscopy. Mol Biol Cell. 2015 26(22):4075-86.
3: Fouquet C, Gilles JF, Heck N, Dos Santos M, Schwartzmann R, Cannaya V, Morel MP, Davidson RS, Trembleau A, Bolte S. Improving axial resolution in confocal microscopy with new high refractive index mounting media. PLoS One. 2015 10(3):e0121096.

NucBlue™ Live ReadyProbes™ Reagent (Invitrogen™)

Hoechst 33342 is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to DNA. With NucBlue® Live ReadyProbes® Reagent we have formulated this classic stain in a room temperature-stable solution that is provided in a convenient-to-use dropper bottle. Just tip and drip two drops per ml to stain your cells.

• No need to dilute, weigh, or pipette
• Convenient dropper bottle—just use two drops per mL
• Stable at room temperature—keep handy at your scope or cell culture area
• Excited by UV light and emits blue fluorescence at 460 nm when bound to DNA nm

See other ReadyProbes® reagents for cell staining
See other nuclear stains for imaging

Cell imaging applications
The spectral properties of Hoechst 33342 (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5'-bi-1H-benzimidazole), including a large Stokes shift, make it ideal for use with green (Alexa Fluor® 488, FITC, GFP) and red (Alexa Fluor® 594, Texas Red®, rhodamine, mCherry, mKate-2) fluorophores in multicolor experiments. Because of its high affinity to DNA, Hoechst 33342 is also frequently used in cell counting, cell cycle, and cell replication studies to distinguish condensed nuclei in apoptotic cells, for cell-cycle studies in combination with Click-iT® EdU or BrdU staining, as a nuclear segmentation tool in high content imaging analysis, and to sort cells based on their DNA content..

Suggestions for use
NucBlue® Live Cell Stain may be added directly to cells in full media, or buffer solutions.
• In most cases 2 drops/ml and an incubation of 15 to 30 minutes will give bright nuclear staining; however, optimization may be needed for some cell types, conditions, and applications. In such cases simply add more, or fewer, drops until the optimal staining intensity is obtained. In most cases, staining intensity increases with time if cells are not washed prior to imaging.
• NucBlue® Live Cell Stain is excited by UV light at 360 nm when bound to DNA, with an emission maximum at 460 nm. It is detected through a blue/cyan filter, such as a DAPI filter, blue GFP filters, or the Semrock BrightLine® Alexa Fluor® 350 Dye filter set.

NucBlue™ Fixed Cell ReadyProbes™ Reagent (Invitrogen™)

DAPI is a commonly used nuclear counterstain for fixed cells that emits blue fluorescence when bound to DNA. With NucBlue® Fixed Cell ReadyProbes® Reagent, we have formulated a high-purity form of this classic stain in a room temperature-stable solution that is provided in a convenient-to-use dropper bottle. Just tip and drip two drops per ml to stain your cells.

• No need to dilute, weigh, or pipette
• Convenient dropper bottle—just use two drops per mL
• Stable at room temperature—keep handy at your scope or cell culture area• DAPI is excited with UV light and detected through a blue/cyan filter

See other ReadyProbes® reagents for cell staining
See other nuclear stains for imaging

Cell imaging applications
DAPI is a classic fluorescent dye used extensively for nuclear staining of fixed cells. In fluorescence microscopy, DAPI is excited with UV light and detected through a blue/cyan filter (Figure 1)..

Suggestions for use
In most cases 2 drops/ml of NucBlue® Fixed Cell Stain and an incubation of 15 to 30 minutes will produce bright nuclear staining; however, optimization may be needed for some cell types, conditions, and applications. In such cases, simply add more, or fewer, drops until the optimal staining intensity is obtained.
• NucBlue® Fixed Cell Stain is excited by UV light at 360 nm when bound to DNA, with an emission maximum at 460 nm. It is detected through a blue/cyan filter, such as a DAPI filter, blue GFP filters, or the Semrock BrightLine® Alexa Fluor® 350 Dye filter set.
• As a preferred blue nuclear stain in fixed cell imaging experiments, NucBlue® Fixed Cell Stain is ideal for use with antibody-based applications.

Molecular Probes® Cell Imaging Kit (Invitrogen™)

Fluorescence imaging protocols are sometimes complicated and tedious, with more time spent preparing samples and reagents than acquiring images and data. To help researchers focus more on biology and get results faster, we have developed a set of imaging reagents that emphasize user friendliness and workflow convenience.

The Molecular Probes® Cell Imaging Kit contains the following products:

NucBlue™ Live ReadyProbes™ Reagent (Cat. No. R37605)
NucRed™ Live 647 ReadyProbes™ Reagent (Cat. No. R37106)
ReadyProbes™ Cell Viability Imaging Kit, Blue/Green (Cat. No. R37609)
Goat Anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor™ 594 conjugate (Cat. No. R37117)
Goat Anti-Mouse lgG Secondary Antibody, Alexa Fluor™ 488 conjugate (Cat. No. R37120)
Image-iT™ FX Signal Enhancer ReadyProbes™ Reagent (Cat. No. R37107)

ReadyProbes™ Cell Viability Imaging Kit, Blue/Red (Invitrogen™)

ReadyProbes® Cell Viability Imaging Kit (Blue/Red) is a ready-to-use kit that can quickly and easily determine the viability of cells. Just add 2 drops each of room temperature, stable NucBlue® Live reagent (Hoechst 33342) and propidium iodide to 1 mL of cell growth media, then determine viability by counting total vs dead cells. NucBlue® Live reagent stains the nuclei of all the cells and can be detected using a standard DAPI filter. Propidium iodide stains only the nuclei of cells with compromised plasma membrane integrity and is detected with a standard TRITC/RFP (orange) filter set. This kit is appropriate for fluorescence microscopy, fluorescence micro plate readers, and flow cytometry.

NucBlue® Live reagent: stains the nuclei of all cells; detected with a standard DAPI filter (excitation/emission maxima: 360/460 nm)
Propidium iodide: stains the nuclei of dead cells with compromised plasma membrane; detected with standard TRITC/RFP (orange) filter set (excitation/emission maxima: 535/617 nm)
See other ReadyProbes® reagents for cell staining
Learn more about other assays for cell viability

Suggestions for use
• NucBlue® Live reagent and propidium iodide can be added directly to cells in full growth media or a compatible buffer solution.
• In most cases, 2 drops/mL and an incubation time of 5 to 30 minutes will give bright nuclear staining; however, optimization may be needed for some cell types, conditions, and applications. In such cases, simply add more or fewer drops until the optimal staining intensity is obtained.

ReadyProbes™ Cell Viability Imaging Kit, Blue/Green (Invitrogen™)

ReadyProbes® Cell Viability Imaging Kit (Blue/Green) is a ready-to-use assay that can quickly and easily determine the viability of cells. Just add 2 drops each of room temperature, stable NucBlue® Live reagent (Hoechst 33342) and NucGreen® Dead reagent to 1 mL of cell growth media, then determine viability by counting total vs dead cells. NucBlue® Live reagent stains the nuclei of all the cells and can be detected with a standard DAPI filter. NucGreen® Dead reagent stains only the nuclei of cells with compromised plasma membrane integrity and is detected using a standard FITC/GFP (green) filter set. This kit is appropriate for fluorescence microscopy, fluorescence microplate readers, and flow cytometry.

NucBlue® Live reagent: stains the nuclei of all cells; detected with a standard DAPI filter (excitation/emission maxima: 360/460 nm)
NucGreen® Dead reagent: stains only the nuclei of dead cells with compromised plasma membranes; detected with standard FITC/GFP (green) filter set (excitation/emission maxima: 504/523 nm)
See other ReadyProbes® reagents for cell staining
Learn more about other assays for cell viability

Suggestions for use
• NucBlue® Live and NucGreen® Dead reagents may be added directly to cells in full growth media or a compatible buffer solution.
• In most cases, 2 drops/mL and an incubation time of 5 to 30 minutes will give bright nuclear staining; however, optimization may be needed for some cell types, conditions, and applications. In such cases, simply add more or fewer drops until the optimal staining intensity is obtained.

Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit (Invitrogen™)

The Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit enables optimal image-based analysis of four key markers of human pluripotent stem cells (PSCs): OCT4, SOX2, SSEA4, and TRA-1-60. This high performance immunocytochemistry (ICC) kit includes a complete set of primary and secondary antibodies, a nuclear DNA stain, and premade buffers for an optimized staining experiment. To characterize pluripotent stem cells using only two markers, we recommend us of the Pluripotent Stem Cell Immunocytochemistry Kit (OCT4, SSEA4) or the Pluripotent Stem Cell Immunocytochemistry Kit (SOX2, TRA-1-60).

With the Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit, you can:
• Confirm expression of key pluripotent stem cell markers with highly specific antibodies
• Get more information per sample by measuring more than one marker at a time
• Generate beautiful multiplexed images with minimal effort
• Save time and avoid cell loss by minimizing unnecessary wash steps in your staining protocol
• Process samples with confidence using a complete and optimized set of ICC reagents

Confirm Expression of Key PSC Markers
The antibodies included in the kit have been carefully selected and validated for high performance in the ICC analysis of PSCs, whether cultured under feeder-dependent or feeder-free media systems. OCT4 and SOX2 are critical nuclear markers, while SSEA4 and TRA-1-60 are important cell surface markers.

Get More Information Per Sample
The antibody combinations in the kit have been designed to allow for the specific and simultaneous assessment of two markers at a time (e.g., OCT4 & SSEA4 and SOX2 & TRA-1-60) along with nuclear DNA staining to help save time and precious sample.

Generate Beautiful Multiplexed Images
Powered by the superior performance of bright, photostable Alexa Fluor® dyes, this kit enables the acquisition of beautiful multiplexed images of PSC marker expression using commonly available blue, green, and orange/red filter sets.

Minimize Unnecessary Wash Steps
The streamlined method recommended for this kit eliminates unnecessary wash steps found in standard ICC protocols to help save time and decrease the likelihood of cell loss during the staining protocol, especially when using glass coverslips or chamber slides. The only washes required are after incubation with primary and secondary antibodies.

Process Samples with Confidence
An optimized set of premade fixation, permeabilization, blocking, and wash buffers allows you to focus on answering scientific questions rather than spending time preparing reagents and determining compatibility.

What You Get
Easily scaled to any culture plate, dish, or slide format, the Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit comes with reagents sufficient to stain 40 samples using a 50 uL staining volume. (See kit content details below.)

Pluripotent Stem Cell Immunocytochemistry Kit (OCT4, SSEA4) (Invitrogen™)

The Pluripotent Stem Cell Immunocytochemistry Kit (OCT4, SSEA4) enables optimal image-based analysis of two key markers of human pluripotent stem cells (PSCs): OCT4 and SSEA4. This high performance immunocytochemistry (ICC) kit includes a complete set of primary and secondary antibodies, a nuclear DNA stain, and premade buffers for an optimized staining experiment. To characterize PSCs with other marker combinations, we recommend the use of the Pluripotent Stem Cell 4-Marker Immunocytochemistry Kit or the Pluripotent Stem Cell Immunocytochemistry Kit (SOX2, TRA-1-60).

With the Pluripotent Stem Cell Immunocytochemistry Kit (OCT4, SSEA4), you can:
• Confirm expression of key pluripotent stem cell markers with highly specific antibodies
• Get more information per sample by measuring more than one marker at a time
• Generate beautiful multiplexed images with minimal effort
• Save time and avoid cell loss by minimizing unnecessary wash steps in your staining protocol
• Process samples with confidence using a complete and optimized set of ICC reagents

Confirm Expression of Key PSC Markers
The antibodies included in the kit have been carefully selected and validated for high performance in the ICC analysis of PSCs, whether cultured under feeder-dependent or feeder-free media systems. OCT4 is a critical nuclear marker, while SSEA4 is an important cell surface marker.

Get More Information Per Sample
The antibodies in the kit have been designed to allow for the specific and simultaneous assessment of OCT4 and SSEA4 along with nuclear DNA staining to help save time and precious sample.

Generate Beautiful Multiplexed Images
Powered by the superior performance of bright, photostable Alexa Fluor® dyes, this kit enables the acquisition of beautiful multiplexed images of PSC marker expression using commonly available blue, green, and orange/red filter sets.

Minimize Unnecessary Wash Steps
The streamlined method recommended for this kit eliminates unnecessary wash steps found in standard ICC protocols to help save time and decrease the likelihood of cell loss during the staining protocol, especially when using glass coverslips or chamber slides. The only washes required are after incubation with primary and secondary antibodies.

Process Samples with Confidence
An optimized set of premade fixation, permeabilization, blocking, and wash buffers allows you to focus on answering scientific questions rather than spending time preparing reagents and determining compatibility.

What You Get
Easily scaled to any culture plate, dish, or slide format, the Pluripotent Stem Cell Immunocytochemistry Kit (OCT4, SSEA4) comes with reagents sufficient to stain 40 samples using a 50 uL staining volume. (See kit content details below.)