24.1 Reference Standards and Antifade Reagents

Transcription

1 24.1 Reference Standards and Antifade Reagents To obtain accurate and reproducible results from fluorescence imaging applications, it is essential to maximize the performance of the optical system. Careful calibration and instrumentation adjustment are required for high-precision imaging of fluorescent probes, particularly in multicolor applications that involve multiple exposures, repetitive scans or three-dimensional sectioning. Molecular Probes offers a variety of microsphere reference standards designed to facilitate adjustment and calibration of both conventional fluorescence microscopes and confocal laser-scanning microscopes. Our Reference Dye Sampler Kit (R-14782) provides ready-made stock solutions of five extensively characterized fluorescence standards for use in spectrofluorometers and fluorescence microplate readers. Because imaging sensitivity depends not only on a well-adjusted instrument, but also on the intensity and stability of the fluorescence signal, Molecular Probes has developed several antifade reagents to minimize photobleaching of fluorescently labeled specimens. Our popular FluoCells prepared microscope slides (Figure 24.1, Figure 24.16, Figure 24.17, Figure 24.18, Figure 24.19, Figure 24.20) are extremely useful products for educational and commercial demonstrations. 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, F-7234) Green-fluorescent and dark red-fluorescent ring stains (15 µm, F-7240) Green fluorescent, orange fluorescent and dark red-fluorescent ring stains (6 µm, F-14806; 15 µm, F-7235) 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, F-14808; 15 µm, F-7237) FocalCheck Fluorescent Microspheres FocalCheck Ring-Stained Fluorescent Microspheres Our patented FocalCheck fluorescent microspheres are specifically designed for examining the alignment, sensitivity and stability of confocal laser-scanning microscopes. 1 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 24.2, Figure 24.3). We refer to this proprietary staining procedure as ring staining, so as to differentiate it from routine staining throughout the bead. 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 24.4). Moreover, because the dyes are localized within the bead and therefore protected from environmental factors, the Focal- Check microspheres are brighter and much more photostable than conventional surface-stained beads. Figure 24.1 FluoCells prepared slide #3 (F-24630) contains a section of mouse kidney stained with a combination of fluorescent dyes. Alexa Fluor 488 wheat germ agglutinin (W-11261), a green-fluorescent lectin, was used to label elements of the glomeruli and convoluted tubules. The filamentous-actin prevalent in glomeruli and the brush border were stained with red-fluorescent Alexa Fluor 568 phalloidin (A-12380). Finally, the nuclei were counterstained with the blue-fluorescent DNA stain DAPI (D-1306, D-3571, D-21490). This image is a composite of three micrographs acquired using filter sets appropriate for fluorescein, tetramethylrhodamine and DAPI. Section

2 A B C Figure 24.2 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, fluorescence emitted 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. Figure 24.3 Images from confocal laser-scanning microscope optical crosssectioning of our 15 µm FocalCheck microspheres that have a dark red-fluorescent ring stain with a green-fluorescent stain throughout the bead (F-7239). The left panel provides a clear visual representation of poor instrument alignment. Correct image registration has been achieved in the right panel, where the dark-red ring is aligned with the green disk. Image contributed by Paulette Brunner, Keck Imaging Center, University of Washington, and Yu-Zhong Zhang, Molecular Probes, Inc. Figure 24.4 Normalized excitation spectra of the dyes contained in the Focal- Check 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. 880 Chapter 24 Accessories and Resources

3 Green-fluorescent ring stain with dark red-fluorescent stain throughout (15 µm, F-7238) Orange-fluorescent ring stain with blue-fluorescent stain throughout (15 µm, F-7236) Dark red-fluorescent ring stain with green-fluorescent stain throughout (6 µm, F-14807; 15 µm, F-7239, Figure 24.3) 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 24.2). FocalCheck Thin-Ring Fluorescent Microspheres Our FocalCheck Thin-Ring Fluorescent Microspheres Kit (F-14791) 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 laserscanning microscope. The FocalCheck Thin-Ring Fluorescent Microspheres Kit 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 redfluorescent (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 (F-24633) or the 15 µm (F-24634) bead size: FocalCheck beads with green-fluorescent, orange-fluorescent and dark red-fluorescent ring stains FocalCheck beads with green-fluorescent ring stain and bluefluorescent stain throughout the bead FocalCheck beads with dark red-fluorescent ring stain and green-fluorescent stain throughout the bead MultiSpeck, TetraSpeck and Constellation Fluorescent Microspheres multicolor applications. These uniform, multiply stained microspheres are useful for co-localizing 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 (M-7901) exhibit three relatively distinct emission bands blue, green and red throughout every particle (Figure 24.5). 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: A suspension of MultiSpeck multispectral microspheres A 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 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 1,2 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 (Figure 24.6), 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: 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 0.1 µm (T-7279) 0.2 µm (T-7280) 0.5 µm (T-7281) 1.0 µm (T-7282) 4.0 µm (T-7283) Section

4 Each of these products provides a 0.5 ml suspension sample of TetraSpeck microspheres that is sufficient for about 100 slides. We offer the TetraSpeck Fluorescent Microspheres Sampler Kit (T-7284) and the TetraSpeck Fluorescent Microspheres Size Kit (T-14792). 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 have 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 has a mixture of all five sizes. Figure 24.5 Multiple exposures of a prepared slide from our MultiSpeck Multispectral Fluorescence Microscopy Standards Kit (M-7901). This kit provides fluorescent MultiSpeck microspheres that exhibit three relatively distinct excitation/emission bands blue, green and red all in the same particle. Thus, the same microsphere can appear a different color depending on the optical filter set used. This photograph was taken through bandpass optical filter sets appropriate for DAPI, fluorescein and Texas Red dyes, with the field of view shifted slightly between exposures. Constellation Microspheres for Imaging Constellation microspheres for imaging (C-14837, Figure 6.3, Figure 24.7) 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. PS-Speck Microscope Point Source Kit The fluorescent microspheres in Molecular Probes PS-Speck Microscope Point Source Kit (P-7220) have a diameter of ± 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 3 (Figure 24.8, Figure 24.9). 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 for the user s convenience. Each suspension provides sufficient material to mount about 100 slides. InSpeck Microscopy Image Intensity Calibration Kits Figure 24.6 Four separate exposures of three TetraSpeck beads (T-7283) photographed using optical filter sets appropriate for DAPI, fluorescein, tetramethylrhodamine and the Texas Red dye. The stage was shifted after each exposure. Note that the same beads appear blue, green, orange or red, depending on the filters used. InSpeck Microscope Image Intensity Calibration Kits provide microsphere standards that generate a series of well-defined fluorescence intensity levels (Figure 24.10) for Figure 24.7 Constellation microspheres (C-14837) provide an assortment of sizes and colors for use in fluorescence microscopy demonstrations. Figure 24.8 One PS-Speck green-fluorescent microsphere (P-7220) used for point-spread function analysis. Shown are individual images, in pseudocolor, taken in x-y planes with 0.25 µm increments in the z-axis between planes. Image contributed by Dr. Regina Armstrong, Uniformed Services University of the Health Sciences, Bethesda, Maryland. Figure 24.9 An orthogonal (x-z) display representing a point-spread function of a particular microscope s optics. The microsphere used is a component of the PS-Speck Microscope Point Source Kit (P-7220). This pseudocolored image was generated electronically from a series of microsphere images taken in x-y planes. Image contributed by Jennifer Kramer, Scanalytics. 882 Chapter 24 Accessories and Resources

5 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, I-7221) InSpeck Green (505/515 nm) Kits (2.5 µm, I-7219; 6 µm, I-14785) InSpeck Orange (540/560 nm) Kits (2.5 µm, I-7223; 6 µm, I-14786) InSpeck Red (580/605 nm) Kits (2.5 µm, I-7224; 6 µm, I-14787) InSpeck Deep Red (633/660 nm) Kit (2.5 µm, I-7225) Each kit includes six separate suspensions of InSpeck fluorescent microspheres with relative fluorescence intensities of 100%, 30%, 10%, 3%, 1% and 0.3% (Figure 24.10), 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. Each suspension provides sufficient material to prepare about 100 slides. Reference Dye Sampler Kit Our Reference Dye Sampler Kit (R-14782) 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 500 diluted working samples for spectrofluorometry. The compositions of the stock solutions are as follows: Figure Flow cytometric analysis of the beads in the 6 µm InSpeck Green Microscope Image Intensity Calibration Kit (I-14785). The microspheres have nominal relative fluorescence intensities of 100%, 30%, 10%, 3%, 1%, 0.3%. For each lot, actual relative intensities are determined by flow cytometry and printed on the product labels. Quinine sulfate in 0.1 M sulfuric acid (H 2 SO 4 ) (Figure 24.11) Fluorescein in dimethylsulfoxide (DMSO) (Figure 24.12) 5-Carboxytetramethylrhodamine in DMSO (Figure 24.13) Sulforhodamine 101 in DMSO (Figure 24.14) Nile blue perchlorate in DMSO (Figure 24.15) Spectroscopic data for the five standards are summarized in Table FluoCells Prepared Microscope Slides In response to requests from educators and instrument manufacturers, Molecular Probes offers FluoCells prepared microscope slides that contain multi-labeled cell preparations for observation by epifluorescence or confocal microscopy. Figure Absorption and fluorescence emission spectra of quinine sulfate, dihydrate, a component of the Reference Dye Sampler Kit (R-14782), in 0.5 M sulfuric acid. FluoCells prepared slide #1 2 (F-14780, Figure 24.16) shows bovine pulmonary artery endothelial cells (BPAEC) stained with MitoTracker Red CMXRos for labeling mitochondria, BODIPY FL phallacidin for labeling F-actin and DAPI for labeling the nucleus. Table 24.1 Spectroscopic data for components of Molecular Probes Reference Dye Sampler Kit. Component Solvent Abs (nm) * Em (nm) * QY Quinine sulfate Water Fluorescein Water CTMR ** Methanol Sulforhodamine 101 Ethanol Nile blue Ethanol * Approximate absorbance (Abs) and fluorescence emission (Em) maxima. Fluorescence quantum yield at 22 C. Standard values in 0.5 M H 2 SO 4. Standard values in 0.1 M NaOH. ** CTMR = Carboxytetramethylrhodamine. Figure Absorption and fluorescence emission spectra of fluorescein, a component of the Reference Dye Sampler Kit (R-14782), in 0.1 M sodium hydroxide. Section

6 Figure Absorption and fluorescence emission spectra of 5-carboxytetramethylrhodamine (5-TAMRA, a component of the Reference Dye Sampler Kit (R-14782)), in methanol. FluoCells prepared slide #2 2 (F-14781, Figure 24.17) again contains BPAEC, but this time stained with Texas Red-X phalloidin for labeling F-actin, mouse monoclonal anti α-tubulin in conjunction with BODIPY FL goat anti mouse IgG for labeling microtubules and DAPI for labeling the nucleus. FluoCells prepared slide #3 (F-24630; Figure 24.1, Figure 24.18, Figure 24.19) 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 (F-24631, Figure 24.20) 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. The multicolor staining in these cell preparations results in stunning, publicationquality images. They 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, specific staining patterns for at least six months from the date of purchase. ProLong and SlowFade Antifade Kits 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, SlowFade and SlowFade Light Antifade Kits, which have been proven to increase the photostability of Figure Absorption and fluorescence emission spectra of sulforhodamine 101, a component of the Reference Dye Sampler Kit (R-14782), in ethanol. Figure Absorption and fluorescence emission spectra of nile blue, a component of the Reference Dye Sampler Kit (R-14782), in ethanol. Figure FluoCells prepared slide #1 (F-14780) showing bovine pulmonary artery endothelial cells (BPAEC) that have been labeled with MitoTracker Red CMXRos (M-7512) for mitochondria, and then fixed, permeabilized and stained with BODIPY FL phallacidin (B-607) for F-actin and DAPI (D-1306, D-3571, D-21490) for nuclei. 884 Chapter 24 Accessories and Resources

7 Figure FluoCells prepared slide #2 (F-14781), which shows bovine pulmonary artery endothelial cells (BPAEC) that have been stained with an anti β-tubulin mouse monoclonal antibody in conjunction with BODIPY FL goat anti mouse IgG (B-2752) for labeling microtubules, Texas Red-X phalloidin (T-7471) for labeling F-actin and DAPI (D-1306, D-3571, D-21490) for labeling nuclei. Figure FluoCells prepared slide #3 (F-24630) contains a mouse kidney section stained with a combination of fluorescent dyes. Greenfluorescent Alexa Fluor 488 wheat germ agglutinin (W-11261) was used to label elements of the glomeruli and convoluted tubules. The filamentous actin was stained with red-fluorescent Alexa Fluor 568 phalloidin (A-12380). Finally, the nuclei were stained with the blue-fluorescent DAPI (D-1306, D-3571, D-21490). This pseudocolored image was acquired on a Zeiss confocal microscope located at the Institute of Neuroscience, University of Oregon. Figure FluoCells prepared slide #3 (F-24630) containing a 16 µm cryostat section of mouse kidney stained with green-fluorescent Alexa Fluor 488 wheat germ agglutinin (W-11261), red-fluorescent Alexa Fluor 568 phalloidin (A-12380) and blue-fluorescent DAPI (D-1306, D-3571, D-21490). The image represents an optical section obtained by simultaneous two-photon excitation of all three dyes at 797 nm using a Bio-Rad Radiance 2100 multi-photon microscope system. Image acquired at the D Microscopy of Living Cells Course, University of British Columbia, Vancouver, Canada, by John Jordan, Bio-Rad Laboratories; and Iain Johnson, Molecular Probes, Inc. Figure FluoCells prepared slide #4 (F-24631) contains a section of mouse intestine stained with a combination of fluorescent stains. Alexa Fluor 350 wheat germ agglutinin (W-11263) is a blue-fluorescent lectin that was used to stain the mucus of goblet cells. The filamentous actin prevalent in the brush border was stained with red-fluorescent Alexa Fluor 568 phalloidin (A-12380). Finally, the nuclei were stained with SYTOX Green nucleic acid stain (S-7020). This image is a composite of three digitized images obtained with filter sets appropriate for fluorescein, DAPI and tetramethylrhodamine. Section

8 Figure The relative photobleaching rates of fluorescein and Texas Red fluorophores with buffer alone (top series) or after treatment with the ProLong antifade reagent (bottom series). The numbers above each pair of frames represents the duration of continuous excitation in seconds. BPAE cells were fixed, permeabilized and labeled with Texas Red-X phalloidin (T-7471), which labels F-actin, and with a mouse monoclonal anti β-tubulin and fluorescein goat anti mouse IgG (F-2761), which label microtubules. Images were acquired at appropriate wavelengths using a cooled CCD camera. 2 Figure Bleaching profiles of A) fluorescein and B) Texas Red dye conjugates in cell samples. In these photobleaching experiments, human epithelium (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 P-7481), 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. Figure Bovine pulmonary arterial epithelial (BPAE) cells were labeled with fluorescein phalloidin (F-432), which labels filamentous actin, and placed under constant illumination on the microscope with a FITC filter set using a 60 objective. Images were acquired at one-second intervals for 30 seconds. Under these illumination conditions, fluorescein photobleached to about 12% of its initial value in 30 seconds in PBS (left), but stayed at the initial value under the same illumination conditions when mounted using the reagents in the ProLong Antifade Kit (right, P-7481). Figure The fluorescence intensity of fluorescein as a function of illumination time, under the following conditions: in the presence of the SlowFade Light antifade reagent (in Kit S-7461; green), in the presence of the SlowFade antifade reagent (in Kit S-2828; red) and in the absence of an antifade reagent (blue). In these experiments, we added free fluorescein directly to a solution and then examined the mixture in a capillary tube using a 20 lens. In real samples such as cells and tissues, we find that the local environment influences the bleaching rate, yielding results that are sometimes different from those shown here. 886 Chapter 24 Accessories and Resources

9 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 highspeed film or high-efficiency detectors. ProLong Antifade Kit The ProLong Antifade Kit (P-7481, Figure 24.21) outperforms all other commercially available antifade reagents. Each ProLong Antifade Kit contains: ProLong antifade reagent powder ProLong mounting medium A protocol for mounting samples Specimens mounted using the ProLong Antifade Kit exhibit little or no quenching of the fluorescent signal of most dyes. Bovine pulmonary arterial epithelial (BPAE) cells labeled with fluorescein phalloidin (F-432) 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 (Figure 24.22). As shown in Figure 24.23, the ProLong Antifade Kit provides more fluorescence output than a popular p-phenylenediamine containing antifade reagent 4 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 (Table 24.6) becomes noticeably brighter upon addition of the ProLong reagents. The ProLong Antifade Kit reagents also inhibit the fading of tetramethylrhodamine, as well as the fading of DNAbound nucleic acid stains such as DAPI, propidium iodide and YOYO-1 (Section 8.1), again without significantly quenching the fluorescence of these dyes. The compatibility of the ProLong Antifade Kit reagents with a multitude of dyes and dye complexes makes it an especially valuable tool for multicolor analysis procedures such as multiplex fluorescence in situ hybridization (FISH, Section 8.5). 5 SlowFade and SlowFade Light Antifade Kits Our original SlowFade formulation (S-2828) was designed to reduce the fading rate of fluorescein to almost zero. Because it provides nearly constant emission intensity from fluorescein, the SlowFade 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. The SlowFade reagent 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, the original SlowFade formulation substantially quenches the fluorescence of fluorescein and almost completely quenches that of the Cascade Blue and Alexa Fluor 350 fluorophores. To overcome this limitation, Molecular Probes researchers have developed the SlowFade Light Antifade Kit (S-7461). The antifade formulation in our SlowFade Light Antifade Kit slows fluorescein s fading rate by about fivefold without significantly reducing fluorescein s initial fluorescence intensity (Figure 24.24), thereby dramatically increasing the signal-to-noise ratio in photomicroscopy. Moreover, quenching of the Cascade Blue, Alexa Fluor 350, tetramethylrhodamine and Texas Red dyes is minimal. In fact, the SlowFade Light antifade reagent reduces the fading rate of the Cascade Blue fluorophore to almost zero, while decreasing its emission intensity by only about 30%. Each SlowFade or SlowFade Light Antifade Kit contains: SlowFade (in Kit S-2828) or SlowFade Light (in Kit S-7461) antifade reagent in 50% (v/v) glycerol, ready to use and sufficient for at least 200 coverslip-size experiments 2 Concentrated SlowFade or SlowFade Light 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 both SlowFade formulations A detailed protocol for mounting samples We have also formulated the SlowFade and SlowFade Light Antifade Kits (S-24635, S-24636) to include the blue-fluorescent nuclear counterstain DAPI (Section 8.6). These kits permit simultaneous staining and protection of the stained sample from photobleaching. The SlowFade Antifade Kits are especially designed for those who want a quick and easy solution to photobleaching. Unlike the ProLong Antifade Kit, the SlowFade Kits require no mixing. Simply pre-wash fluorescently labeled specimens in the equilibration buffer provided and then apply the mounting medium directly from the convenient applicator bottle. References 1. Appl Immunohistochem Mol Morphol 7, 156 (1999); 2. Methods 18, 447 (1999); 3. Biophys J 80, 2455 (2001); 4. J Histochem Cytochem 41, 1833 (1993); 5. Cytometry 32, 163 (1998). Product List 24.1 Fluorescence Microscopy and Fluorometry Reference Standards and Antifade Reagents Cat # Product Name Unit Size C Constellation microspheres for imaging *mixture of assorted sizes and colors*... 3 ml F FluoCells prepared slide #1 *BPAE cells with MitoTracker Red CMXRos, BODIPY FL phallacidin, DAPI*... each F FluoCells prepared slide #2 *BPAE cells with mouse anti-α-tubulin, BODIPY FL goat anti-mouse IgG, Texas Red -X phalloidin, DAPI*... each F FluoCells prepared slide #3 *mouse kidney section with Alexa Fluor 488 WGA, Alexa Fluor 568 phalloidin, DAPI*... each Section

10 Product List 24.1 Fluorescence Microscopy and Fluorometry Reference Standards and Antifade Reagents continued Cat # Product Name Unit Size F FluoCells prepared slide #4 *mouse intestine section with Alexa Fluor 350 WGA, Alexa Fluor 568 phalloidin, SYTOX Green*... each F FocalCheck Fluorescent Microspheres Kit, 6 µm *mounted on slides*... 1 kit F FocalCheck Fluorescent Microspheres Kit, 15 µm *mounted on slides*... 1 kit F FocalCheck microspheres, 6 µm, fluorescent dark-red ring stain/green throughout ml F FocalCheck microspheres, 6 µm, fluorescent green/orange/dark-red ring stains ml F FocalCheck microspheres, 6 µm, fluorescent green ring stain/blue throughout ml F-7234 FocalCheck microspheres, 15 µm, fluorescent blue/orange ring stains ml F-7239 FocalCheck microspheres, 15 µm, fluorescent dark-red ring stain/green throughout ml F-7240 FocalCheck microspheres, 15 µm, fluorescent green/dark-red ring stains ml F-7235 FocalCheck microspheres, 15 µm, fluorescent green/orange/dark-red ring stains ml F-7237 FocalCheck microspheres, 15 µm, fluorescent green ring stain/blue throughout ml F-7238 FocalCheck microspheres, 15 µm, fluorescent green ring stain/dark red throughout ml F-7236 FocalCheck microspheres, 15 µm, fluorescent orange ring stain/blue throughout ml F FocalCheck Thin-Ring Fluorescent Microspheres Kit, 1.0 µm *three suspensions*... 1 kit I-7221 InSpeck Blue (350/440) Microscope Image Intensity Calibration Kit, 2.5 µm... 1 kit I-7225 InSpeck Deep Red (633/660) Microscope Image Intensity Calibration Kit, 2.5 µm... 1 kit I-7219 InSpeck Green (505/515) Microscope Image Intensity Calibration Kit, 2.5 µm... 1 kit I InSpeck Green (505/515) Microscope Image Intensity Calibration Kit, 6 µm... 1 kit I-7223 InSpeck Orange (540/560) Microscope Image Intensity Calibration Kit, 2.5 µm... 1 kit I InSpeck Orange (540/560) Microscope Image Intensity Calibration Kit, 6 µm... 1 kit I-7224 InSpeck Red (580/605) Microscope Image Intensity Calibration Kit, 2.5 µm... 1 kit I InSpeck Red (580/605) Microscope Image Intensity Calibration Kit, 6 µm... 1 kit M-7901 MultiSpeck Multispectral Fluorescence Microscopy Standards Kit *in suspension*... 1 kit P-7481 ProLong Antifade Kit... 1 kit P-7220 PS-Speck Microscope Point Source Kit *blue, green, orange and deep red fluorescent beads*... 1 kit R Reference Dye Sampler Kit *five 1 mm solutions, 1 ml each*... 1 kit S-2828 SlowFade Antifade Kit... 1 kit S SlowFade Antifade Kit with DAPI... 1 kit S-7461 SlowFade Light Antifade Kit... 1 kit S SlowFade Light Antifade Kit with DAPI... 1 kit T-7284 TetraSpeck Fluorescent Microspheres Sampler Kit... 1 kit T TetraSpeck Fluorescent Microspheres Size Kit *mounted on slides*... 1 kit T-7279 TetraSpeck microspheres, 0.1 µm, fluorescent blue/green/orange/dark red ml T-7280 TetraSpeck microspheres, 0.2 µm, fluorescent blue/green/orange/dark red ml T-7281 TetraSpeck microspheres, 0.5 µm, fluorescent blue/green/orange/dark red ml T-7282 TetraSpeck microspheres, 1.0 µm, fluorescent blue/green/orange/dark red ml T-7283 TetraSpeck microspheres, 4.0 µm, fluorescent blue/green/orange/dark red ml 24.2 Flow Cytometry Reference Standards Flow cytometers are designed to perform quantitative measurements on individual cells and other particles with high precision, speed and accuracy. As with all high-performance instrumentation, flow cytometers must be calibrated frequently to ensure accuracy and reliability. The stability, uniformity and reproducibility of our fluorescent microsphere products make them ideal reference standards for flow cytometry. Our flow cytometry reference standards exhibit superior stability and greater uniformity than other commercially available microparticles, and they are accompanied by a full one-year warranty. Known numbers of fluorescent microspheres can also be added to samples to facilitate the estimation of cell numbers, 1 as in our Bacteria Counting Kit 2 4 (B-7277, see below). Unfortunately, because of high variability in quantum yields of dyes once bound to an antibody, heterogeneity of antibody labeling, problems with stoichiometry and accessibility in binding to targets, improper calibration procedures and several other factors, bead standards containing a known number of fluorophores per bead do not necessarily provide accurate information on the number of ligands bound to a cell. 5 AlignFlow and AlignFlow Plus Flow Cytometry Alignment Beads Due to advances in flow cytometric instrumentation and the development of fluorescent probes, a scientist can now perform multiparameter analyses on biological samples. In order to ensure accurate and reproducible results, flow cytometers should be checked at least daily for proper performance. Molecular Probes AlignFlow and AlignFlow Plus flow cytometry alignment beads permit the calibration of a flow cytometer s laser(s), optics and stream flow without wasting valuable and sensitive experimental material. These fluorescently stained, polystyrene microspheres are 888 Chapter 24 Accessories and Resources