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Histogene™ Staining Solution (Applied Biosystems™)

This staining solution is for use with the ARCTURURS® HistoGene® LCM Frozen Section Staining Kit.

Designed as a fast-penetrating stain, it provides excellent contrast while preserving nucleic acid integrity and retaining low-abundance mRNAs in frozen tissue sections intended for laser capture microdissection (LCM).

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

Histogene™ Refill Kit, includes dehydration chemicals & stain only (Applied Biosystems™)

This kit contains only the dehydration chemicals and stain used with the ARCTURUS® HistoGene® LCM Frozen Section Staining Kit. Designed as a fast-penetrating stain, this kit provides excellent contrast while preserving nucleic acid integrity and retaining low-abundance mRNAs.

Key product features:
• Optimized process—provide superior staining while preserving RNA
• Comprehensive kit—simplify tissue staining and dehydration
• High-quality RNA yield—retain low-abundance mRNA and maintain RNA integrity
• Versatile application—obtain high-quality RNA from many tissue types
• Modular design—use with other ARCTURUS® products to produce superior microarray data

Provide Superior Staining while Preserving Nucleic Acids
HistoGene® Staining Solution has been developed by ARCTURUS® to stain tissues for LCM subsequently used as sources of nucleic acids. It is a fast-penetrating stain that provides good contrast by differential staining of nuclei (purple) and cytoplasm (light pink). By minimizing the exposure of tissues to water where nucleases may be activated, HistoGene® stain helps to preserve nucleic integrity that may be otherwise compromised when using longer staining protocols.

Retain Low-abundance mRNA
RT-PCR analysis of specific genes from cells captured from tissues processed using the HistoGene® LCM Frozen Section Staining Kit shows retention of both low-abundance mRNA and higher-abundance species. The mRNA profile of HistoGene® samples appears free of degradation.

Maintain RNA Integrity
Tissues prepared for LCM using HistoGene® LCM Frozen Section Staining Kit reagents, supplies and instructions yield high quality RNA.

Obtain High-quality RNA from Many Tissue Types
ARCTURUS® has validated the HistoGene® Kit by examining RNA integrity on many tissue types. All tissues tested to date using the HistoGene® Kit have yielded quality RNA. Validated tissue types include (Table 1):

Part of the Arcturus® System for Microgenomics
The HistoGene® LCM Frozen Section Staining Kit is part of the ARCTURUS® Complete System of Microgenomics and is designed to seamlessly work together to produce high quality expression microarrays. With the PicoPure® RNA Isolation Kit, you can recover a high yield of RNA from as few as 10 cells in a minimal volume. The RiboAmp® RNA Amplification Kit can amplify picograms of your RNA sample into micrograms of amplified RNA (aRNA) that is ready for labeling and hybridization to microarrays. ARCTURUS® instruments and kits increase the reliability and reproducibility of gene expression studies.

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

Histogene™ LCM Immunofluorescence Staining Kit, includes staining components only (Applied Biosystems™)

The ARCTURUS® HistoGene® LCM Immunofluorescence Staining Kit is designed to retrieve high-quality nucleic acids from immunofluorescently-stained, frozen tissue using a quick 15-minute process. The kit provides reagents and slides for convenient and reliable immunoflourescent staining as well as protocols that are streamlined and optimized to maintain nucleic acid quality.

Key product features are:
• Simplified staining—retrieve high-quality RNA from simplified fluorescent target cell labeling
• Efficient preservation—quick 15-minute process preserves mRNA
• Cold sample protection—chill samples for intact RNA
• Exceptional specificity—benefit from brilliant high-contrast label intensity
• Maximized compatibility—use with many tissue types

Simplified Fluorescent Target Cell Labeling
Identify target cells for Laser Capture Microdissection (LCM) and gene expression analysis using antigen markers to highlight specific surface or intracellular proteins. The HistoGene® LCM Immunofluorescence Staining Kit is specifically designed to retrieve high-quality RNA from immunofluorescently stained frozen tissue. The staining kit provides reagents and slides for convenient and reliable immunoflourescent staining as well as protocols that are streamlined and optimized to maintain nucleic acid quality (Figure 1).

Quick 15-minute Process Preserves RNA
The HistoGene® LCM Immunofluorescence Kit works in less than 15 minutes (only 5 minutes in aqueous environment). The proprietary staining buffer from ARCTURUS® minimizes nucleic acid degradation. Standard immunofluorescence labeling kits typically take over 90 minutes to process samples, compromising the nucleic acid integrity with conditions sub-optimal for single-stranded nucleic acid stability. Electrophoresis and RT-PCR of RNA isolated after using the HistoGene® LCM Immunofluorescence Kits confirm that high-quality RNA is retrieved from microdissected samples (Figures 2 & 3).

Chill Samples for Intact RNA
To increase RNA yield and help ensure its quality, the HistoGene® Cold Block keeps up to four tissue section slides and several tubes containing buffer and antibody solutions chilled while using the HistoGene® LCM Immunofluorescence Kit. The HistoGene® Cold Block is designed for use with the CoolSafe triple density polysterene cooler (Cat. # CSF-Box) and -10°C CoolBrick (Cat. # BRIK-1520) from Diversified Biotech (Figure 4).

Obtain Intact RNA from Many Tissue Types
Our scientists have examined RNA integrity after using the HistoGene® Immunofluorescence Kits on many tissue types with several antibodies. All tissue tested using the HistoGene® Immunofluorescence Kits have yielded high-quality RNA. Tissue⁄antibody combinations validated include:

Get Brilliant High-contrast Label Intensity
The HistoGene® LCM Immunofluorescence Kit employs a biotin-avidin system with Cy3 which leads to exceptionally good staining intensity and specificity. The kit is used with a primary biotinylated monoclonal antibody to an antigen of choice provided by the user. Cy3-conjugated streptavidin is included in the kit (Figure 5).

Part of the ARCTURUS® Systems for Microgenomics
The HistoGene® LCM Immunofluorescence Kit is validated as part of the Arcturus® Complete System for Microgenomics, an integrated solution for utilizing small quantities of RNA for gene expression analysis. Use the HistoGene® LCM Immunofluorescence Kit to immunofluorescently stain your samples. Use the ArcturusXT™ Laser Capture Microdissection instrument to capture pure cell populations of your target cells. The PicoPure® RNA Isolation Kit lets you maximize recovery of RNA from even small numbers of cells. The RiboAmp® RNA Amplification Kit can amplify picograms of your RNA sample into micrograms of amplified RNA (aRNA) that is ready for labeling and hybridization to microarrays. ARCTURUS® instruments and kits increase the reliability and reproducibility of gene expression studies.

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

Vybrant™ Apoptosis Assay Kit #5, Hoechst 33342/Propidium Iodide (Invitrogen™)

The Vybrant Apoptosis Assay Kit #5 provides a rapid and convenient assay for apoptosis based on fluorescence detection of the compacted state of the chromatin in apoptotic cells. This kit contains ready-to-use solutions of blue-fluorescent Hoechst 33342 dye, which stains the condensed chromatin of apoptotic cells more brightly than the chromatin of nonapoptotic cells, and red-fluorescent propidium iodide dye, which stains dead cells.

Dead Cell Apoptosis Kit with Annexin V PE and SYTOX™ Green, for flow cytometry (Invitrogen™)

This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to the orange fluorescent phycobiliprotein R-PE, and dead cells using SYTOX™ Green nucleic acid stain. After treatment with both probes, apoptotic cells show orange fluorescence, dead cells show green fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished in the 530/30 nm and 585/42 nm bandpass filters with a 488 nm laser flow cytometer.

View a selection guide for all apoptosis assays for flow cytometry.

eBioscience™ Mouse Regulatory T Cell Staining Kit #2 (Invitrogen™)

The FJK-16s antibody reacts with mouse/rat Foxp3 also known as FORKHEAD BOX P3, SCURFIN, and JM2; cross reactivity of this antibody to other proteins has not been determined. Foxp3 a 49-55 kDa protein, is a member of the forkhead/winged-helix family of transcriptional regulators, and was identified as the gene defective in ‘scurfy’ (sf) mice. Constitutive high expression of Foxp3 mRNA has been shown in CD4+CD25+ regulatory T cells (Treg cells), and ectopic expression of Foxp3 in CD4+CD25- cells imparts a Treg phenotype in these cells.

Immunoblotting with FJK-16s antibody has mapped the epitope to amino acids 75-125 of the mouse Foxp3 protein. In the human, this region has been shown to be alternatively spliced at the mRNA level. Both the alternatively-spliced and non-spliced isoforms are present in the CD4+CD25+ subset of lymphocytes. Preliminary RT-PCR experiments have not revealed this alternatively-spliced isoform in mouse splenocytes, suggesting different gene regulation in the mouse and human.

Intracellular staining of mouse splenocytes with FJK-16s using the mouse Foxp3 staining sets and protocol reveals approximately 2% of total splenocytes in the C57Bl/6 strain and approximately 3-5% in the BALB/c mouse strain. Multicolor flow cytometric analysis demonstrates approximately 90% of the CD4+CD25+ cells and 4% of the CD4+CD25- cells staining with FJK-16s. B220+, CD11b+, CD11c+, and Ly6G/Gr-1+ cells do not show significant co-staining with FJK-16s. These data are consistent with a recent report which follows expression of Foxp3 using a GFP knock-in (Fontenot et al, 2005).

FJK-16s cross-reacts with rat Foxp3 This has been demonstrated by intracellular staining of Foxp3 and flow cytometry of rat splenocytes using the same method and reagents as used for mouse tissue. Please note that the CD4 and CD25 antibodies included in this kit only recognize the mouse antigens. For staining rat tissue, please use (CD4 FITC, Cat. No. 11-0040 and CD25 PE, Cat. No. 12-0390)

The anti-mouse Foxp3 Staining Set has been formulated and optimized for the staining of mouse splenocytes with the FJK-16s monoclonal antibody.

Not included:
Isotype controls for anti-CD4 (rat IgG2a, Cat. No. 11-4321) and anti-CD25 (rat IgG1, Cat. No. 12-4301)

Host
Rat

Isotype
IgG2a

Reactivity/Species
Mouse

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

Amplex™ Red Sphingomyelinase Assay Kit (Invitrogen™)

The Amplex® Red Sphingomyelinase Assay Kit provides a sensitive, rapid, and simple fluorometric method for detecting very low concentrations of sphingomyelinase using a fluorescence microplate reader or fluorometer.

See our complete line of Fluorescence Microplate assays.

• Detects sphingomyelinase activity levels as low as 80 µU/mL in a 200 µL assay volume
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference

In this enzyme-coupled assay, sphingomyelinase activity is monitored indirectly using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red reagent), a sensitive fluorogenic probe for hydrogen peroxide. Sphingomyelinase hydrolyses the sphingomyelin to yield ceramide and phosphorylcholine. Alkaline phosphatase is added, which hydrolyzes phosphorylcholine to form choline. The choline is then oxidized by choline oxidase to form betaine and hydrogen peroxide. In the presence of horseradish peroxidase, the hydrogen peroxide reacts with Amplex® Red reagent in a 1:1 stoichiometric ratio to generate the highly fluorescent product resorufin.

Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples.

Use Amplex® Red Assays for a Broad Range of Investigations
A wide variety of validated Amplex® Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex® UltraRed Reagent (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex® Red/UltraRed Stop Reagent (Cat. No. A33855). The Amplex® Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours. Custom assay design and packaging are also available.

Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI) (Invitrogen™)

The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide is a flow cytometry kit used to measure early apoptosis by detecting phosphatidyl serine expression and membrane permeability.

View a selection guide for all apoptosis assays for flow cytometry.

Superior brightness
Unlike other Annexin V kits that have lower protein concentrations or purification levels, the Annexin V Alexa Fluor™ 488 conjugate is optimized for flow cytometry to provide the largest separation between apoptotic and live cells. The Alexa Fluor™ 488 dye is a superior green-fluorescent dye with a spectrum similar to fluorescein (FITC).

High binding efficiency
Annexin V conjugates are made from a highly purified cys-annexin, which leads to higher binding efficiency, resulting in highly accurate characterization of the apoptotic process.

Multi-parametric
Many publications require a minimum of two different ways to identify that cells are apoptotic. This multi-parametric kit detects phosphatidyl serine (PS) on the cytoplasmic surface of the cell membrane and membrane integrity using propidium iodide.

How it works
When cells are stained with Annexin V and propidium iodide, apoptotic cells expressing PS show green fluorescence, which can be detected in the FITC channel, and low red fluorescence. Dead or necrotic cells show bright red fluorescence and no green fluorescence, while live cells show no green or red fluorescence.

CellTrace™ Far Red Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ Far Red Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP
• Simple, robust staining protocol

View a selection guide for all CellTrace™ Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ Far Red dye enables the visualization of six or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. This kit enables consistent, homogeneous staining results with very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace™ Far Red stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye offers a consistent signal, even after several days in a cell culture environment. CellTrace™ Far Red dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The red excitation at 630 nm and emission at 661 nm of CellTrace™ Far Red dye make it ideal for multiplexing due to the limited spectral overlap with other common dyes (Alexa Fluor™ 488, FITC, and RPE) and fluorescent proteins (Green Fluorescent Protein (GFP) and mCherry).

Simple, robust staining protocol
The CellTrace™ Far Red Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

Dead Cell Apoptosis Kit with Annexin V APC and SYTOX™ Green, for flow cytometry (Invitrogen™)

This flow cytometry product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to red laser-excited allophycocyanin, and dead cells using SYTOX™ Green nucleic acid stain. Apoptotic cells are detected by annexin V binding to externalized phosphatidylserine, and late apoptotic and necrotic cells have compromised membranes that permit SYTOX™ Green stain access to cellular nucleic acids. After exposing a cell population to APC annexin V and SYTOX™ Green stain, live cells show little or no fluorescence, apoptotic cells show red fluorescence and very little green fluorescence, and late apoptotic cells show a higher level of red and orange fluorescence. These populations can easily be distinguished using a flow cytometer that has both 488 nm and 633 nm excitation sources (an argon-ion laser and a HeNe laser).

View a selection guide for all apoptosis assays for flow cytometry.

ABC Peroxidase Standard Staining Kit (Thermo Scientific™)

Thermo Scientific Pierce ABC Staining Kits include reagents for the avidin-biotin complex (ABC) technique, a highly sensitive method for immunohistochemical detection with biotinylated secondary antibodies.

Pierce ABC Staining Kits are highly sensitive, produce very low background staining and have rapid avidin-biotin interactions. Standard Kits contain only the two essential reagents (avidin and biotinylated HRP) can be used with nearly any species of biotinylated secondary antibody. The Ultra-Sensitive ABC Peroxidase Staining Kits are approximately five times more sensitive than the regular-senstivity kit, enabling more dilute primary antibodies to be used while producing equal staining intensity. Ultra-Sensitive Kits are offered as a Standard Kit and as Complete Kits, which contain biotinylated secondary antibodies and appropriate block serum for use with mouse or rabbit antibodies, respectively.

Related Products
Ultra-Sensitive ABC Peroxidase Standard Staining Kit
Ultra-Sensitive ABC Peroxidase Mouse IgG Staining Kit
Ultra-Sensitive ABC Peroxidase Rabbit IgG Staining Kit

eBioscience™ Annexin V Apoptosis Detection Kit APC (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reported Application
Flow Cytometric Analysis

Amplex™ Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen™)

The Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit provides a sensitive and simple method for detecting glutamic acid/glutamate oxidase activity using a fluorescence microplate reader or fluorometer.

See our complete line of Fluorescence Microplate assays.

• Detect concentrations as low as 10 nM L-glutamic acid or 40 µU/mL of purified L-glutamate oxidase
• Format allows for multiple time point measurements
• Designed for minimal autofluorescence interference

The Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit provides an ultrasensitive method for detecting glutamic acid or for continuously monitoring glutamate oxidase activity using a fluorescence microplate reader or fluorometer. In the assay, L-glutamic acid is oxidized by glutamate oxidase to produce α-ketoglutarate, NH3, and hydrogen peroxide. L-alanine and L-glutamate–pyruvate transaminase are included in the reaction to regenerate L-glutamic acid by transamination of α-ketoglutarate, resulting in multiple cycles of the initial reaction and a significant amplification of the hydrogen peroxide produced. The hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex® Red reagent) in a 1:1 stoichiometric ratio in a reaction catalyzed by horseradish peroxidase (HRP) to generate the highly fluorescent product resorufin.

Because resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, there is little interference from autofluorescence in most biological samples.

Use Amplex® Red Assays for a Broad Range of Investigations
A wide variety of validated Amplex® Red assays are available for studying cell signaling and lipids, neurobiology, inflammation and immune function, and metabolism. We also offer Amplex® UltraRed Reagent (Cat. No. A36006), a second-generation reagent providing greater sensitivity and brighter fluorescence, and the Amplex® Red/UltraRed Stop Reagent (Cat. No. A33855). The Amplex® Red/UltraRed Stop Reagent provides convenience and control by allowing the fluorescence signal-generating reaction to be terminated at a user-determined time point. After addition of the stop reagent, the fluorescence signal remains stable for at least three hours. Custom assay design and packaging are also available.

Staphylococcus aureus (Wood strain without protein A) BioParticles™, Alexa Fluor™ 488 conjugate (Invitrogen™)

The Molecular Probes® BioParticles® product line consists of a series of fluorescently labeled, heat- or chemically killed bacteria and yeast in a variety of sizes, shapes, and natural antigenicities. These fluorescent BioParticles® products have been employed to study phagocytosis by fluorescence microscopy, quantitative spectrofluorometry, and flow cytometry.

We offer E. coli (K-12 strain), S. aureus (Wood strain, without protein A) and zymosan (S. cerevisiae) BioParticles® conjugates covalently labeled with a variety of different fluorophores (special care has been taken to remove free dye after conjugation). Unlike the fluorescence of fluorescein-labeled BioParticles® conjugates, which is partially quenched in acidic environments, the fluorescence of the Alexa Fluor®, BODIPY® FL, tetramethylrhodamine and Texas Red® dye conjugates is uniformly intense over the pH range from 3 to 10.

BioParticles Specifications:
• Label (Ex/Em): Alexa Fluor® 488 (~495/519 nm)
• Particle: S. aureus (Wood strain, without protein A)
Opsonizing reagent available


Using BioParticles Products
BioParticles® conjugates are provided as lyophilized powders. There are approximately 3 x 108 E. coli or S. aureus particles per mg solid and approximately 2 x 107 zymosan particles per mg solid. BioParticles® conjugates can be reconstituted in the buffer of your choice for use in phagocytosis assays. The fluorescence of BioParticles® conjugates that are bound to the surface of the cell (but not internalized) can be quenched by ethidium bromide, trypan blue, or other quenchers. In addition to cellular applications, fluorescent BioParticles® conjugates may be effective as flow cytometry calibration references when sorting bacteria and yeast mutants. These small particles may also be useful references for light scattering studies because their sizes and shapes differ in characteristic ways.

Find More BioParticles® Products
We offer a large range of dye-labeled and unlabeled E. coli (K-12 strain), S. aureus (Wood strain, without protein A), and zymosan (S. cerevisiae) BioParticles® products. Find out about these products and their applications by reviewing Probes for Following Receptor Binding and Phagocytosis—Section 16.1 in the Molecular Probes® Handbook.

For pH-sensitive endocytosis assays, see our pHrodo™ BioParticles® conjugates.

For Research Use Only. Not for human or animal therapeutic or diagnostic use.

Click-iT™ EdU Alexa Fluor™ 647 Imaging Kit (Invitrogen™)

The Click-iT® EdU Alexa Fluor® 647 Imaging Kit is a superior alternative to traditional proliferation assays that is optimized for fluorescence microscopy applications. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol.

• Simple—works the first time, every time, in less time
• Efficient—no denaturation steps or harsh treatment required
• Content-rich results—better preservation of cell morphology, antigen structure, and DNA integrity
• Consistent—not dependent on variable antibody lots for detection

The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently. The harsh treatment can also adversely effect sample integrity and quality, which makes co-staining with other antibodies challenging.

Superior Proliferation Methodology
The Click-iT® EdU Alexa Fluor® 647 Imaging Kit provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT® EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT® EdU detection reagent to gain access to the DNA. As a consequence, the Click-iT® EdU imaging kit is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.

This kit is optimized for fluorescence microscopy applications; visit the Click-iT® technology area of our website for kits designed for high-content imaging, fluorescence microplate, or flow cytometry platforms.

Learn more about Click-iT technology >
Find more tools for image-based detection of proliferating cells >

Notes:
The Click-iT® assay can be used on cells in culture or in vivo following EdU administration by feeding or injection methods.
The Click-iT® assay can be used with BrdU in dual pulse experiments by using the anti-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.
The Click-iT® technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed cell labeling.