Fluorescence Microscopy Reference Standards and Antifade Reagents - Section 23.1

FocalCheck Ring-Stained Fluorescent Microspheres

Our Patented FocalCheck fluorescent microspheres (FocalCheck Fluorescent Microsphere Standards) are specifically designed for examining the alignment, sensitivity and stability of confocal laser-scanning microscopes. They are particularly useful for confirming the optical sectioning thickness (Z-resolution) in three-dimensional imaging applications. These polystyrene beads — available in 6 µm and 15 µm diameters — have been treated using a proprietary method in which a fluorescent dye is allowed to penetrate to only a limited depth within the microsphere. The resulting beads have a well-defined dye layer that, when viewed in cross section in the confocal laser-scanning microscope, appears as a fluorescent ring of varying dimensions depending on the focal plane (Figure 23.1, ). We refer to this proprietary staining procedure as ring staining to differentiate it from routine staining throughout the bead.

Figure 23.1 Confocal laser-scanning microscope optical cross-sectioning and alignment with FocalCheck microspheres. A) Serial optical sectioning from top to bottom along the z-axis of ring-stained microspheres reveals a continuous pattern of disc-to-ring-to-disc images. B) The diameter of the fluorescent ring (or disc) seen is dependent on the depth of the optical focal plane. C) In the confocal laser-scanning microscope, separate light paths exist for UV and visible wavelengths. Also, emitted fluorescence is detected by different photomultipliers. Proper optical alignment may be obtained with either of two types of FocalCheck microspheres. For example, the microspheres with an orange ring stain that are blue-fluorescent throughout the bead allow UV/visible wavelength alignment in three dimensions upon aligning the orange ring with the blue disc. Focal alignment is also possible simultaneously in three colors by aligning the green, orange and dark red rings of the FocalCheck microspheres containing fluorescent green/orange/dark red ring stains.

FocalCheck microspheres are available in a variety of color configurations provided by five different fluorescent stains:

• Blue (365/430 nm)
• Green (505/515 nm)
• Orange (560/580 nm)
• Red (575/600 nm)
• Dark red (660/680 nm)

The excitation/emission maxima of the different stains are well matched to the laser sources and optical filters commonly used in confocal laser-scanning microscopy and are especially useful in testing and aligning confocal laser-scanning microscopes with multiple laser lines and detection channels (Figure 23.3). Moreover, because the dyes are localized within the bead and therefore protected from environmental factors, the FocalCheck microspheres are brighter and much more photostable than conventional surface-stained beads.

Figure 23.3 Normalized excitation spectra of the dyes contained in the FocalCheck microspheres. Emission lines of several commonly used laser excitation sources are superimposed on the dyes' excitation spectra to illustrate the wide range of usage of these beads as calibration references. Ar = Argon-ion laser. Kr–Ar = Krypton–argon laser. He–Ne = Helium–neon laser.

FocalCheck products are available in seven different color configurations, including three suspensions that contain microspheres exhibiting ring stains of two or three different fluorescent colors:

• Blue-fluorescent and orange-fluorescent ring stains (15 µm, F7234)
• Green-fluorescent and dark red–fluorescent ring stains (15 µm, F7240)
• Green-fluorescent, orange-fluorescent and dark red–fluorescent ring stains (6 µm, F14806; 15 µm, F7235)

We also supply four suspensions that contain microspheres exhibiting a ring stain of one fluorescent color combined with a stain of a second fluorescent color throughout the bead:

• Green-fluorescent ring stain with blue-fluorescent stain throughout (6 µm, F14808; 15 µm, F7237)
• Green-fluorescent ring stain with dark red–fluorescent stain throughout (15 µm, F7238)
• Orange-fluorescent ring stain with blue-fluorescent stain throughout (15 µm, F7236)
• Dark red–fluorescent ring stain with green-fluorescent stain throughout (6 µm, F14807; 15 µm, F7239, )

The sharp ring stains exhibited by the FocalCheck microspheres produce a striking visual representation of instrument misalignment or other aberrations, making them ideal as reference standards for confocal laser-scanning microscopy. Correct image registration is indicated when the multiple ring images of the ring-stained FocalCheck beads (or the ring and disk images of the combination ring-stained and stained-throughout FocalCheck beads) are perfectly coincident in all dimensions (Figure 23.1).

FocalCheck Thin-Ring Fluorescent Microspheres

Our FocalCheck Thin-Ring Fluorescent Microspheres Kit (F14791) contains smaller-diameter microspheres that have spectral and imaging features similar to those of our 6 µm and 15 µm FocalCheck microspheres. Because we prepare these 1.0 µm beads using fluorescent stains that are restricted to the surface only, they exhibit sharper and thinner fluorescent ring patterns when viewed in cross section with a confocal laser-scanning microscope. The FocalCheck Thin-Ring Fluorescent Microspheres Kit (FocalCheck Thin-Ring Fluorescent Microspheres) contains three 200 µL suspensions of 1.0 µm beads. Each suspension contains beads with a different color configuration:

• Green-fluorescent (495/515 nm) and red-fluorescent (575/600 nm) ring stains
• Green-fluorescent (495/515 nm) ring stain with dark red–fluorescent (660/680 nm) stain throughout the bead
• Red-fluorescent (575/600 nm) ring stain with blue-fluorescent (365/430 nm) stain throughout the bead

FocalCheck Microspheres Pre-Mounted on Microscope Slides

In addition to the bead suspensions described above, we offer FocalCheck microspheres pre-mounted on microscope slides. The FocalCheck Fluorescent Microsphere Kits feature mounted samples of three different color configurations, in either the 6 µm (F24633) or the 15 µm (F24634) bead size:

• FocalCheck beads with green-fluorescent, orange-fluorescent and dark red–fluorescent ring stains
• FocalCheck beads with green-fluorescent ring stain and blue-fluorescent stain throughout the bead
• FocalCheck beads with dark red–fluorescent ring stain and green-fluorescent stain throughout the bead

FocalCheck Fluorescence Microscope Test Slides

FocalCheck fluorescence microscope test slides #1, #2, and #3 are specifically designed for calibrating fluorescence microscope systems and evaluating system and filter performance:

• FocalCheck fluorescence microscope test slide #1 (F36909) is ideal for routine checking and calibration of confocal and widefield fluorescence microscopes (Contents of FocalCheck fluorescence microscope tests slide #1 - Table 23.1).
• FocalCheck fluorescence microscope test slide #2 (F36913) provides a robust, reproducible method of evaluating the performance of spectral imaging systems, as well as the ability to discriminate closely overlapping spectra. The slide consists of 6 µm–diameter microspheres labeled with a series of spectrally overlapping dyes (Contents of FocalCheck fluorescence microscope tests slide #2 - Table 23.2).
• FocalCheck fluorescence microscope test slide #3 (F36914) is useful for basic evaluation of filter performance and as a general practice slide for fluorescence microscopy and digital imaging (Contents of FocalCheck fluorescence microscope tests slide #1 - Table 23.3).

The slides each contain 10 sample areas coated with proprietary fluorescent microspheres designed specifically for microscopy applications. The microspheres are mounted in optical cement (refractive index ~1.52) for maximal stability and are intended for use on all fluorescence microscope systems.

FocalCheck Fluorescent Microspheres for Testing Spectral Separation

Molecular Probes has prepared four FocalCheck Fluorescent Microspheres Kits for testing spectral separation on the Zeiss META system and other spectral imaging systems. These microspheres are stained with two different fluorescent dyes that appear similar in color by eye but are sufficiently different to be resolved by linear-unmixing techniques. When linear-unmixing data-processing algorithms are applied, the dyes are shown to be spectrally distinct and spatially separated — one appears only within the outer ring and the other appears throughout the microsphere (). These 6 µm, dual-stained microspheres are provided mounted on a microscope slide in each of the following kits:

• FocalCheck DoubleGreen Fluorescent Microspheres Kit (F36905), with a green-fluorescent (500/512 nm) ring dye and a slightly longer-wavelength green-fluorescent (512/525 nm) core dye
• FocalCheck DoubleOrange Fluorescent Microspheres Kit (F36906), with an orange-fluorescent (532/552 nm) ring dye and a slightly longer-wavelength orange-fluorescent (545/565 nm) core dye

For generating reference spectra, these kits also contain two additional slides containing microspheres stained uniformly with each of the individual dyes.

Our unique MultiSpeck and TetraSpeck fluorescent microspheres greatly facilitate the adjustment and calibration of conventional fluorescence microscopes, confocal laser-scanning microscopes and associated image-processing equipment for multicolor applications. These uniform, multiply stained microspheres are useful for colocalizing and focusing different wavelengths of light in the same optical plane, as well as for checking multicolor image resolution, magnification and sensitivity.

MultiSpeck Multispectral Fluorescence Microscopy Standards Kit

The 4 µm MultiSpeck microspheres in our MultiSpeck Multispectral Fluorescence Microscopy Standards Kit (M7901) exhibit three relatively distinct emission bands — blue, green and red — throughout every particle (). The spectral characteristics (excitation/emission peaks at 365/405 nm, 520/525 nm and 580/600 nm) are compatible with optical filter sets designed for commonly used blue, green and red fluorophores (e.g., DAPI, fluorescein and rhodamine or Texas Red dyes or their Alexa Fluor dye counterparts). The MultiSpeck beads can be used as external references for comparing images collected with different optics, on different instruments and in different laboratories, as well as for monitoring routine day-to-day variations in instrument performance. Furthermore, because a single multispectral microsphere will appear as different colors depending on the optical filters used for observation, these beads can be used to assess image registration in two and three dimensions, allowing the researcher to accurately determine the spatial relationships of different labels in a multiparameter experiment. The MultiSpeck Multispectral Fluorescence Microscopy Standards Kit contains:

• Suspension of MultiSpeck multispectral microspheres
• Mixed suspension of separately stained blue-, green- and red-fluorescent microspheres, which exhibit the same three excitation/emission bands as the multispectral microspheres but in separate beads
• Mounting medium
• A slide-mounting protocol (Multispeck Multispectral Fluorescence Microscopy Standards Kit)

Both suspensions are provided at a ready-to-use density and can be mounted on slides or incorporated into an experimental sample. Each kit supplies a sufficient amount of material for ~50 slide preparations using either of the two bead suspensions provided.

TetraSpeck Fluorescent Microspheres and Sampler Kits

Our TetraSpeck fluorescent microspheres expand the multispectral strategy introduced with the MultiSpeck beads in two important ways. First, the TetraSpeck beads have been stained throughout with a mixture of four different fluorescent dyes, yielding four well-separated excitation and emission peaks (). The excitation/emission maxima of the dyes are 365/430 nm (blue), 505/515 nm (green), 560/580 nm (orange) and 660/680 nm (dark red). Second, these microspheres are available in five nominal sizes (actual bead diameters are indicated on the product labels), spanning the range from subresolution to nearly cell-size particles:

• 0.1 µm (T7279)
• 0.2 µm (T7280)
• 0.5 µm (T7281)
• 1.0 µm (T7282)
• 4.0 µm (T7283)

Each of these products provides a 0.5 mL suspension sample of TetraSpeck microspheres that is sufficient for about 100 slides (TetraSpeck Fluorescent Microsphere Standards). We offer the TetraSpeck Fluorescent Microspheres Sampler Kit (T7284) and the TetraSpeck Fluorescent Microspheres Size Kit (T14792). The TetraSpeck Fluorescent Microspheres Sampler Kit consists of separate suspension samples of our 0.1 µm, 0.5 µm and 4.0 µm TetraSpeck beads, each sufficient for preparing about 20 slides. The TetraSpeck Fluorescent Microspheres Size Kit contains six microscope slides; five slides with a mounted sample of the 0.1 µm–, 0.2 µm–, 0.5 µm–, 1.0 µm– or 4.0 µm–diameter TetraSpeck microspheres, and a sixth slide with a mixture of all five sizes.

Constellation Microspheres for Imaging

Constellation microspheres for imaging (C14837, , ) are 3 mL suspensions of assorted fluorescent microspheres with a variety of sizes and colors. Designed for use in laboratory tutorials and customer training sessions, they provide inexpensive and robust test samples for demonstrating filter switching, focus adjustment and other functional capabilities of fluorescence microscopes. The Constellation microspheres can be stored at room temperature, protected from light.

The fluorescent microspheres in Molecular Probes' PS-Speck Microscope Point Source Kit (P7220) have a diameter of 0.175 ± 0.005 µm, making them ideal subresolution fluorescent sources for calibrating instrument optics. They are particularly useful for measuring the point-spread function required for computational image deconvolution procedures (, ). This kit's four ready-to-use suspensions contain bright monodisperse particles in the following fluorescent colors (and excitation/emission peaks):

• Blue (360/440 nm)
• Green (505/515 nm)
• Orange (540/560 nm)
• Deep red (633/660 nm)

The kit also includes mounting medium and a mounting protocol (PS-Speck Microscope Point Source Kit) for the user's convenience. Each suspension provides sufficient material to mount about 100 slides.

InSpeck Microscope Image Intensity Calibration Kits provide microsphere standards that generate a series of well-defined fluorescence intensity levels (Figure 23.10) for constructing calibration curves and evaluating sample brightness. Most of the kits are offered in a choice of two different microsphere sizes (2.5 µm or 6 µm) and five different fluorescent colors:

• InSpeck Blue (350/440 nm) Kit, (2.5 µm, I7221)
• InSpeck Green (505/515 nm) Kits (2.5 µm, I7219; 6 µm, I14785)
• InSpeck Orange (540/560 nm) Kits (2.5 µm, I7223; 6 µm, I14786)
• InSpeck Red (580/605 nm) Kits (2.5 µm, I7224; 6 µm, I14787)
• InSpeck Deep Red (633/660 nm) Kit (2.5 µm, I7225)

Each kit includes six separate suspensions of InSpeck fluorescent microspheres with relative fluorescence intensities of 100%, 30%, 10%, 3%, 1% and 0.3% (Figure 23.10), covering the range of intensities commonly encountered in microscopy applications. Unstained control beads and mounting medium are also supplied. The aqueous suspensions of microspheres may be applied directly to the sample for calibrating fluorescence intensities or mounted separately in an adjacent well or on another slide (InSpeck Microscope Image Intensity Calibration Kits). Each suspension provides sufficient material to prepare about 100 slides.

Fluorescein NIST-Traceable Standard

The National Institute of Standards and Technology (NIST) chose a high-grade fluorescein synthesized by Molecular Probes to create Standard Reference Material 1932 (SRM 1932), a certified fluorescein solution. Molecular Probes now offers a NIST-traceable fluorescein standard (F36915) that not only meets the stringent criteria established by NIST, but is also directly traceable to SRM 1932. We supply our NIST-traceable fluorescein standard as a calibrated 50 µM solution of fluorescein in 100 mM sodium borate buffer, pH 9.5; under these conditions, fluorescein is completely ionized and is therefore in its most fluorescent form (Figure 20.1, Figure 20.2), exhibiting an extremely high quantum yield of 0.93 (Probes Useful at Near-Neutral pH - Section 20.2).

Figure 20.1 Ionization equilibria of fluorescein.

Figure 20.2 The pH-dependent spectra of fluorescein (F1300): A) absorption spectra, B) emission spectra.

Academic researchers and industry scientists alike can use our NIST-traceable fluorescein standard to assess day-to-day or experiment-to-experiment variation in fluorescence-based instrumentation, as well as to determine the Molecules of Equivalent Soluble Fluorophore (MESF) value for an experimental solution. The MESF value is defined not as the actual number of dye molecules present, but rather as the number of fluorophores that would yield a fluorescence intensity equivalent to that of the experimental solution when analyzed on the same instrument under the same conditions. Consequently, the MESF value is an important tool for characterizing the fluorescence intensity of a solution containing spectrally similar dye molecules attached to antibodies, nucleic acids, microspheres or other substrates that might enhance or diminish the fluorescence. When its pH is carefully matched with that of the experimental solution, our NIST-traceable fluorescein standard can be used for accurate MESF determinations of a wide range of green-fluorescent dye solutions and on an assortment of fluorescence-based instruments.

Reference Dye Sampler Kit

Our Reference Dye Sampler Kit (R14782) provides samples of five extensively characterized fluorescence standards with emission spectra covering the entire visible wavelength range. All five fluorescent standards are supplied as 1 mM stock solutions in 1 mL units, sufficient to prepare approximately 500 diluted working samples for spectrofluorometry (Reference Dye Sampler Kit). The compositions of the stock solutions are as follows:

• Quinine sulfate in 0.1 M sulfuric acid (H₂SO₄) ()
• Fluorescein in dimethylsulfoxide (DMSO) ()
• 5-Carboxytetramethylrhodamine in DMSO ()
• Sulforhodamine 101 in DMSO ()
• Nile blue perchlorate in DMSO ()

Spectroscopic data for the five standards are summarized in Spectroscopic data for components of Molecular Probes' Reference Dye Sampler Kit - Table 23.4.

Ideal for educators and instrument manufacturers, our popular FluoCells prepared microscope slides contain multilabeled cell preparations for observation by epifluorescence or confocal laser-scanning microscopy. The multicolor staining in these cell preparations results in stunning, publication-quality images and lasts through repeated viewings. These slides are especially useful for setting up microscopes and camera systems and for assessing the capabilities of optical filter sets. When stored properly, these permanently mounted specimens will retain their bright and specific staining patterns for at least six months from the date of purchase. We currently offer five different FluoCells prepared microscope slides:

• FluoCells prepared slide #1 (F36924 shows bovine pulmonary artery endothelial cells (BPAEC) stained with MitoTracker Red CMXRos for labeling mitochondria, Alexa Fluor 488 phalloidin for labeling F-actin and DAPI for labeling the nucleus.
• FluoCells prepared slide #2 (F14781, ) again contains BPAEC, but this time stained with Texas Red-X phalloidin for labeling F-actin, mouse monoclonal anti–α-tubulin antibody in conjunction with BODIPY FL goat anti–mouse IgG antibody for labeling microtubules and DAPI for labeling the nucleus.
• FluoCells prepared slide #3 (F24630; , , ) contains a 16 µm cryostat section of a mouse kidney. Green-fluorescent Alexa Fluor 488 wheat germ agglutinin stains the glomeruli and convoluted tubules; red-fluorescent Alexa Fluor 568 phalloidin labels actin, which is especially prevalent in glomeruli and the brush border of proximal convoluted tubules; finally, DAPI stains the nuclei with blue fluorescence.
• FluoCells prepared slide #4 (F24631, ) contains a 16 µm cryostat section of a mouse intestine. Alexa Fluor 350 wheat germ agglutinin labels the mucus of goblet cells with blue fluorescence; the red-fluorescent Alexa Fluor 568 phalloidin labels actin filaments, which are especially prevalent in the brush border of the intestinal mucosa; and SYTOX Green nucleic acid stain labels nuclei with green fluorescence.
• FluoCells prepared slide #6 (F36925, ) contains muntjac skin fibroblast cells stained with a combination of fluorescent stains. Green-fluorescent Alexa Fluor 488 phalloidin labels the prominent filamentous actin in these cells; a mouse monoclonal anti–OxPhos Complex V inhibitor protein antibody in conjunction with the orange-fluorescent Alexa Fluor 555 goat anti–mouse IgG antibody labels mitochondria; far-red–fluorescent TO-PRO-3 nucleic acid stain labels nuclei. Because it contains no blue-fluorescent dyes, this slide is ideal for use with confocal laser-scanning microscopes that rely on non–UV laser light sources.

Loss of fluorescence through irreversible photobleaching processes can lead to a significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration. To minimize photobleaching of experimental samples, we have developed the ProLong, ProLong Gold, SlowFade and SlowFade Gold Antifade Kits and reagents, which have been shown to increase the photostability of many of our fluorophores in fixed cells, fixed tissues and cell-free preparations. The primary function of any antifade reagent is to sustain dye fluorescence, usually by inhibiting the generation and diffusion of reactive oxygen species. Strategies for further maximizing the fluorescence signal in both living and nonliving specimens include reducing the excitation light intensity by using neutral density filters, high–numerical aperture objectives, relatively low magnification, high-quality optical filters and high-speed film or high-efficiency detectors.

ProLong Antifade Kit

The ProLong Antifade Kit (P7481, ) contains our original ProLong antifade reagent, which has proven to effectively enhance the resistance of many different fluorophores to photobleaching. Furthermore, specimens mounted using the ProLong Antifade Kit exhibit little or no quenching of the fluorescent signal of most dyes. Each ProLong Antifade Kit contains:

• ProLong antifade reagent powder
• ProLong mounting medium
• A protocol for mounting samples (ProLong(R) Antifade Kit)

Bovine pulmonary arterial epithelial cells (BPAEC) labeled with fluorescein phalloidin (F432) photobleached to about 12% of the initial value in 30 seconds in PBS, while staying at the initial value under the same illumination conditions when mounted using the ProLong Antifade Kit (). As shown in Figure 23.24, the ProLong Antifade Kit provides more fluorescence output than a popular p-phenylenediamine–containing antifade reagent when used to mount fluorescein-stained HEp-2 cells. Furthermore, fluorescence of the Texas Red dye when viewed through the appropriate Texas Red optical filter (Spectral characteristics and recommended bandpass filter sets for Molecular Probes' dyes - Table 23.11) becomes noticeably brighter upon addition of the ProLong antifade reagent. The ProLong antifade reagent also inhibits the fading of tetramethylrhodamine, as well as the fading of DNA-bound nucleic acid stains such as DAPI, propidium iodide and YOYO-1 (Nucleic Acid Stains - Section 8.1), again without significantly quenching the fluorescence of these dyes. The compatibility of the ProLong antifade reagent with a multitude of dyes and dye complexes makes it an especially valuable tool for multicolor analysis procedures such as multiplexed fluorescence in situ hybridization (FISH, Detecting Nucleic Acid Hybridization - Section 8.5).

Figure 23.24 Bleaching profiles of A) fluorescein and B) Texas Red dye conjugates in cell samples. In these photobleaching experiments, human epithelial (HEp-2) cells were probed with human anti-nuclear antibodies and then developed for visualization with fluorophore-labeled secondary reagents. Identical samples were mounted in ProLong antifade reagent (: in Kit P7481), Product X (+) or medium containing no antifade reagent (). Although these data were normalized, we observed little or no quenching of samples mounted with the ProLong mounting medium.

ProLong Gold Antifade Reagent

ProLong Gold antifade reagent is a new and even more effective version of the ProLong antifade reagent, a component of the ProLong Antifade Kit described above. The ProLong Gold antifade reagent outperforms all other commercially available antifade reagents and causes little or no quenching of the fluorescent signal (Figure 23.25, , ). Furthermore, unlike the reagents in the ProLong Antifade Kit, the ProLong Gold antifade reagent is premixed and ready to use — just add a drop to the preparation and mount. As with our original ProLong antifade reagent, ProLong Gold reagent cures within 24 hours and the sample can be saved for months after mounting. ProLong Gold reagent offers excellent compatibility with a multitude of dyes and dye complexes, making it an especially valuable tool for multicolor applications. The ProLong Gold antifade reagent is available in a single 10 mL bottle (P36930), as well as in a set of five 2 mL bottles (P36934).

As an added convenience, we also offer ProLong Gold antifade reagent containing DAPI, the popular nuclear and chromosome stain that emits blue fluorescence upon binding to DNA. The addition of DAPI in the mounting media eliminates the need for a separate counterstaining step. ProLong Gold antifade reagent with DAPI is available in a single 10 mL bottle (P36931), as well as in a set of five 2 mL bottles (P36935).

SlowFade Antifade Kit

Our original SlowFade antifade formulation (S2828) was designed to reduce the fading rate of fluorescein to almost zero. Because it provides nearly constant emission intensity from fluorescein, this SlowFade antifade reagent is especially useful for quantitative measurements and applications that employ a confocal laser-scanning microscope, in which the excitation intensities can be extreme and prolonged. Use of the SlowFade Antifade Kit can extend the useful fluorescence emission of fluorescein more than 50-fold and can preserve the signal in cell and tissue mounts for up to two years. However, this original SlowFade formulation substantially quenches the fluorescence of fluorescein and almost completely quenches that of the Alexa Fluor 350, Alexa Fluor 405 and Cascade Blue fluorophores.

Each SlowFade Antifade Kit contains:

• SlowFade antifade reagent in 50% (v/v) glycerol, ready to use and sufficient for at least 200 coverslip-size experiments
• Concentrated SlowFade antifade reagent solution, provided for those applications in which glycerol may not be compatible
• Equilibration buffer, which raises the pH of the sample, increasing the protection afforded by the SlowFade antifade formulation
• Detailed protocols for mounting samples (SlowFade(R) Antifade Kits)

SlowFade Gold Antifade Reagent

To overcome the limitations of the original SlowFade antifade reagent, especially with blue fluorophores, Molecular Probes' researchers have developed the SlowFade Gold antifade reagent. The SlowFade Gold antifade formulation slows fluorescein's fading rate by about fivefold without significantly reducing fluorescein's initial fluorescence intensity, thereby dramatically increasing the signal-to-noise ratio in photomicroscopy. Moreover, quenching of the Alexa Fluor 350, Alexa Fluor 405, Cascade Blue, tetramethylrhodamine and Texas Red dyes is minimal. In fact, the SlowFade Gold antifade reagent reduces the fading rate of the Cascade Blue fluorophore to almost zero, while decreasing its emission intensity by only about 30%.

The SlowFade Gold antifade reagent is available in a single 10 mL bottle (S36936), as well as in a set of five 2 mL bottles (S36937). As with the ProLong Gold antifade reagents, we also offer SlowFade Gold antifade reagent containing the blue-fluorescent nuclear counterstain DAPI (Nuclear and Chromosome Counterstaining and Nissl Stains - Section 8.6). SlowFade Gold antifade reagent with DAPI is available in a single 10 mL bottle (S36938), as well as in a set of five 2 mL bottles (S36939). These reagents permit simultaneous nuclear staining and protection of the stained sample from photobleaching.

Unlike the ProLong Gold antifade reagents, the SlowFade Gold antifade reagents do not cure over time so samples can be viewed immediately; however, SlowFade Gold reagents are intended for short-term use (3–4 weeks) only and mounted samples may degrade over time.

Image-iT FX Kits

The Image-iT FX Kits (Image-iT FX Kits - Table 7.6) provide the best secondary detection reagents and supporting materials needed for optimal imaging of fixed cells and tissue sections:

• Alexa Fluor conjugates of goat anti–mouse IgG antibody, goat anti–rabbit IgG antibody or streptavidin deliver superior photostability and brightness (Fluorescence Characteristics of the Alexa Fluor Dyes in the Image-iT FX Kits - Table 7.7, see Secondary Immunoreagents - Section 7.2 for more specific information)
• ProLong Gold antifade reagent reduces photobleaching (Figure 23.25, , )
• Image-iT FX signal enhancer improves the signal-to-noise ratio (, , )
• A sample pack of two CultureWell chambered coverglasses makes sample processing more convenient (Figure 23.35, see Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3 for more details)

Each Image-iT FX Kit provides sufficient materials to perform 50–100 assays. Furthermore, the components of each kit are available separately (Alexa Fluor secondary antibodies, Secondary Immunoreagents - Section 7.2, Summary of Molecular Probes' secondary antibody conjugates - Table 7.1; Alexa Fluor streptavidin, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23; ProLong Gold antifade reagent, P36930; Image-iT FX signal enhancer, I36933; CultureWell chambered coverglasses, C37000, C37005, Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3) for flexibility in experimental design.

Image-iT FX Signal Enhancer

By efficiently blocking nonspecific interactions of a wide variety of fluorescent dyes with cell and tissue constituents, the Image-iT FX signal enhancer (I36933) dramatically improves the signal-to-noise ratio of immunolabeled cells and tissues, allowing clear visualization of targets that would normally be indistinguishable due to background fluorescence (, , ). Background staining seen with fluorescent conjugates of streptavidin (Fluorescent dyes successfully tested with the Image-iT FX signal enhancer * - Table 7.8), goat anti–mouse IgG antibody or goat anti–rabbit IgG antibody is largely eliminated when Image-iT FX signal enhancer is applied to fixed and permeabilized cells prior to staining. Image-iT FX signal enhancer may also effectively prevent nonspecific staining that is typically blocked with 1–2% BSA or 10% serum treatment, in some cases eliminating the need for another step in the staining protocol.