14.5 Fluorescent and Biotinylated Dextrans. Properties of Our Dextran Conjugates. Dextran Size. A Wide Selection of Substituents

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1 Cat # Product Name Unit Size D ,1 -dilinoleyl-3,3,3,3 -tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (FAST DiI solid; DiI 9,12 -C 18 (3), CBS)... 5 mg D ,1 -dilinoleyl-3,3,3,3 -tetramethylindocarbocyanine perchlorate (FAST DiI oil; DiI 9,12 -C 18 (3), ClO 4 )... 5 mg D ,1 -dioctadecyl-5,5 -diphenyl-3,3,3,3 -tetramethylindocarbocyanine chloride (5,5 -Ph 2 -DiIC 18 (3))... 5 mg D ,3 -dioctadecyl-5,5 -di(4-sulfophenyl)oxacarbocyanine, sodium salt (SP-DiOC 18 (3))... 5 mg D ,1 -dioctadecyl-6,6 -di(4-sulfophenyl)-3,3,3,3 -tetramethylindocarbocyanine (SP-DiIC 18 (3))... 5 mg D-275 3,3 -dioctadecyloxacarbocyanine perchlorate ( DiO ; DiOC 18 (3)) mg D ,1 -dioctadecyl-3,3,3,3 -tetramethylindocarbocyanine-5,5 -disulfonic acid (DiIC 18 (3)-DS)... 5 mg D-282 1,1 -dioctadecyl-3,3,3,3 -tetramethylindocarbocyanine perchlorate ( DiI ; DiIC 18 (3)) mg D ,1 -dioctadecyl-3,3,3,3 -tetramethylindocarbocyanine perchlorate ( DiI ; DiIC 18 (3)) *crystalline* mg D ,1 -dioctadecyl-3,3,3,3 -tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt ( DiD solid; DiIC 18 (5) solid) mg D ,1 -dioctadecyl-3,3,3,3 -tetramethylindodicarbocyanine-5,5 -disulfonic acid (DiIC 18 (5)-DS)... 5 mg D-307 1,1 -dioctadecyl-3,3,3,3 -tetramethylindodicarbocyanine perchlorate ( DiD oil; DiIC 18 (5) oil) mg D ,1 -dioctadecyl-3,3,3,3 -tetramethylindotricarbocyanine iodide ( DiR ; DiIC 18 (7)) mg D ,1 -dioleyl-3,3,3,3 -tetramethylindocarbocyanine methanesulfonate ( 9 -DiI) mg L-7781 Lipophilic Tracer Sampler Kit... 1 kit N NeuroTrace CM-DiI tissue-labeling paste mg N NeuroTrace DiD tissue-labeling paste mg N NeuroTrace DiI tissue-labeling paste mg N NeuroTrace DiO tissue-labeling paste mg N NeuroTrace Multicolor Tissue-Labeling Kit *DiO, DiI, DiD pastes, 500 mg each*... 1 kit O-246 octadecyl rhodamine B chloride (R18) mg T-3163 N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM 1-43)... 1 mg T-1111 N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridinium dibromide (RH 414)... 5 mg T-3166 N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM 4-64)... 1 mg T N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM 4-64) *special packaging* x 100 µg T N-(3-trimethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM 5-95)... 1 mg V Vybrant CM-DiI cell-labeling solution... 1 ml V Vybrant DiD cell-labeling solution... 1 ml V Vybrant DiI cell-labeling solution... 1 ml V Vybrant DiO cell-labeling solution... 1 ml V Vybrant Multicolor Cell-Labeling Kit *DiO, DiI, DiD solutions, 1 ml each*... 1 kit 14.5 Fluorescent and Biotinylated Dextrans Dextrans are hydrophilic polysaccharides characterized by their moderate to high molecular weight, good water solubility and low toxicity. They are widely used as both anterograde and retrograde tracers in neurons 1,2 and for numerous other applications. Dextrans are biologically inert due to their uncommon poly-(α-d-1,6-glucose) linkages, which render them resistant to cleavage by most endogenous cellular glycosidases. They also usually have low immunogenicity. Molecular Probes offers almost 100 fluorescent and biotinylated dextran conjugates in several molecular weight ranges. Properties of Our Dextran Conjugates A Wide Selection of Substituents Molecular Probes dextrans are conjugated to biotin or a wide variety of fluorophores, including five of our Alexa Fluor dyes (Table 14.4). In particular, we would like to highlight the dextran conjugates of our Alexa Fluor 488, Oregon Green and Rhodamine Green dyes, which are significantly brighter and more photostable than most fluorescein dextrans. Dextran-conjugated fluorescent indicators for calcium and magnesium ions (Section 20.4) and for ph (Section 21.4) are described with their corresponding ion indicators in other chapters. Dextran Size Molecular Probes dextrans include those with nominal molecular weights (MW) of 3000; 10,000; 40,000; 70,000; 500,000; and 2,000,000 daltons (Table 14.4). Because unlabeled dextrans are polydisperse and may become more so during the chemical processes required for their modification and purification the actual molecular weights present in a particular sample may have a broad distribution. For example, our 3000 MW dextran preparations contain polymers with molecular weights predominantly in the range of ~ daltons, including the dye or other label. Degree of Substitution of Our Dextrans Dextrans from other commercial sources usually have a degree of substitution of 0.2 or fewer dye molecules per dextran molecule for dextrans in the 10,000 MW range. Our dextrans, however, typically contain dyes per dextran in the 3000 MW range, dyes per dextran in the 10,000 MW range, 2 4 dyes Section

2 in the 40,000 MW range and 3 6 dyes in the 70,000 MW range. The actual degree of substitution is indicated on the product s label. If too many fluorophores are conjugated to the dextran molecule, quenching and undesired interactions with cellular components may occur. We have found our degree of substitution to be optimal for most applications, yielding dextrans that are typically much more fluorescent than the labeled dextrans available from other sources, thus permitting lower quantities to be used for intracellular tracing. It has been reported that some commercially available fluorescein isothiocyanate (FITC) dextrans yield spurious results in endocytosis studies because of the presence of free dye or metal contamination. 3,4 To overcome this problem, Molecular Probes removes as much of the free dye as possible by a combination of precipitation, dialysis, gel filtration and other techniques. The fluorescent dextran is then assayed by thin-layer chromatography (TLC) to ensure that it is free of low molecular weight dyes. Molecular Probes prepares several unique products that have two or even three different labels, including our fluoro-ruby, mini-ruby and micro-ruby products, described below. Not all of the individual dextran molecules of these products are expected to have all the substituents, or to be equally fixable, particularly in conjugates of the lowest molecular weight dextrans. Dextran Net Charge and Method of Substitution The net charge on the dextran depends on the fluorophore and the method of preparing the conjugate. Molecular Probes prepares most of its dextrans by reacting a water-soluble amino dextran (D-1860, D-1861, D-1862, D-3330, D-7144) with the succinimidyl ester of the appropriate dye, rather than reacting a native dextran with isothiocyanate derivatives such as FITC. This method provides superior amine selectivity and yields an amide linkage, which is somewhat more stable than the corresponding thioureas formed from isothiocyanates. Except for the Rhodamine Green and Alexa Fluor 488 conjugates, once the dye has been added, the unreacted amines on the dextran are capped to yield a neutral or anionic dextran. In the case of the Rhodamine Green and Alexa Fluor 488 dextrans, the unreacted amines on the dextran are not capped after dye conjugation. Thus, these dextran conjugates may be either neutral, anionic or cationic. The Alexa Fluor, Cascade Blue, lucifer yellow, fluorescein and Oregon Green dextrans are intrinsically anionic, whereas most of the dextrans labeled with the zwitterionic rhodamine B, tetramethylrhodamine and Texas Red dyes are essentially neutral. To produce more highly anionic dextrans, Molecular Probes has developed a proprietary procedure for adding negatively charged groups to the Table 14.4 Molecular Probes selection of dextran conjugates. Label(s) (Absorption/Emission Maxima) * Cascade Blue (400/420) D-7132 D-1976 Lucifer yellow (428/532) D-1825 Alexa Fluor 488 (494/521) D MW 10,000 MW 40,000 MW 70,000 MW 500,000 MW 2,000,000 MW Oregon Green 488 (496/524) D-7170, D-7171 D-7172, D-7173 Fluorescein (494/518) D-3305, D-3306 D-1821, D-1820 D-1844, D-1845 D-1823, D-1822 D-7136 D-7137 Fluorescein + biotin (494/518) D-7156 D-7178 DMNB-caged fluorescein (494/518) D-3310 DMNB-caged fluorescein + biotin (494/518) D-7146 Rhodamine Green (502/527) D-7163 D-7153 BODIPY FL (505/513) D-7168 Oregon Green 514 (511/530) D-7175 D-7176 Tetramethylrhodamine (555/580) D-3307, D-3308 D-1816, D-1817, D-1868 Alexa Fluor 546 (556/573) D Tetramethylrhodamine + biotin (555/580) D-7162 D-3312 Rhodamine B (570/590) D-1824 D-1841 Alexa Fluor 568 (578/603) D Alexa Fluor 594 (590/617) D D-1842 D-1819, D-1818 D-7139 Texas Red (595/615) D-3329, D-3328 D-1828, D-1863 D-1829 D-1830, D-1864 Alexa Fluor 647 (650/668) D Biotin (<300/none) D-7135 D-1956 D-1957 D-7142 Amino (<300/none) D-3330 D-1860 D-1861 D-1862 D-7144 * Approximate absorption and emission maxima, in nm, for conjugates. Lysine-fixable dextrans contain lysines and can therefore be fixed in place with formaldehyde or glutaraldehyde. Fixable dextrans contain free amines (but not lysines) and can be fixed in place with formaldehyde or glutaraldehyde. Absorption and emission maxima are for the conjugate following photolysis. 582 Chapter 14 Fluorescent Tracers of Cell Morphology and Fluid Flow

3 dextran carriers; these products are designated polyanionic dextrans. Dextran Fixability Some applications require that the dextran tracer be subsequently treated with formaldehyde or glutaraldehyde for analysis. 5,6 For these applications, Molecular Probes offers lysine-fixable versions of most of our dextran conjugates of fluorophores or biotin. These dextrans have covalently bound lysine residues that permit dextran tracers to be conjugated to surrounding biomolecules by aldehyde-mediated fixation for subsequent detection by immunohistochemical and ultrastructural techniques. We have also shown that all of our Alexa Fluor dextran conjugates can be fixed with aldehyde-based fixatives. Loading Cells with Dextrans and Subsequent Tissue Processing Unless taken up by an endocytic process, dextran conjugates are membrane impermeant and usually must be loaded by relatively invasive techniques (Table 14.1). As with the lipophilic tracers in Section 14.4, crystals of the dextran conjugates have been successfully loaded by simply placing them directly on some kinds of samples. 7 We have found our Influx pinocytic cellloading reagent (I-14402, Section 14.3, Section 20.8) to be particularly useful for loading dextrans into a variety of adherent and nonadherent cells (Figure 20.94). Sterile filtration of dextran solutions before use with live cells is highly recommended. 8 Biotin and biotinylated biomolecules with molecular weights up to >100,000 daltons are taken up by some plant cells through an endocytic pathway. 9,10 Our lysine-fixable dextrans and Alexa Fluor dextrans can be fixed in place with formaldehyde or glutaraldehyde, allowing subsequent tissue processing, such as sectioning. A protocol has been published for embedding tissues in plastic for high-resolution characterization of neurons filled with lysine-fixable fluorescent dextrans. 11 Fixation of biotinylated or fluorescent dextrans also permits the use of fluorescent- or enzyme-labeled conjugates of avidin and streptavidin (Section 7.6, Table 7.17) or of anti-dye antibodies (Section 7.4, Table 7.13), respectively. These techniques can amplify the signal, which is important for detecting fine structure in sections or for changing the detection mode. 12 We provide antibodies to the Alexa Fluor 488, Cascade Blue, lucifer yellow, fluorescein, BODIPY FL, tetramethylrhodamine and Texas Red fluorophores and to the 2,4-dinitrophenyl (DNP) and nitrotyrosine haptens (Section 7.4). Our antibodies to fluorescein crossreact strongly with the Oregon Green dyes and somewhat with the Rhodamine Green fluorophore, and our anti-tetramethylrhodamine and anti Texas Red antibodies crossreact with tetramethylrhodamine, Lissamine rhodamine B, Rhodamine Red and Texas Red dyes. Molecular Probes ELF 97 Immunohistochemistry Kit and ELF 97 Cytological Labeling Kits (E-6600, E-6603; Section 6.3) and several of our TSA (Tyramide Signal- Amplification) Kits (Section 6.2) can be utilized directly with biotinylated dextrans or combined with antibodies to the fluorophore-labeled dextrans to further amplify the signal, making it possible to detect ultrafine structures accessible to these dextran conjugates. The NANOGOLD and Alexa Fluor FluoroNanogold conjugates of secondary antibodies (Section 7.3) and streptavidin (Section 7.6) can be utilized to allow detection of labeled dextrans in fixed-cell preparations by light microscopy or, following silver enhancement with the LI Silver Enhancement Kit (L-24919, Section 7.3), by electron microscopy. Photoconversion of neurons labeled with lysine-fixable fluorescent dextrans in the presence of diaminobenzidine (DAB) using our Diaminobenzidine (DAB) Histochemistry Kits (Section 7.3, Section 7.6) can be used to produce electron-dense products for electron microscopy 13 (see Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents in Section 1.5). Electrondense products can also be generated from peroxidase or colloidal gold conjugates of avidin, streptavidin or anti-dye antibodies. 14 Neuronal Tracing with Dextrans Fluorescent and biotinylated dextrans are routinely employed to trace neuronal projections. Dextrans can function efficiently as anterograde or retrograde tracers, 2 depending on the study method and tissue type used. Active transport of dextrans occurs only in live, not fixed tissue. 15 Comparative studies of rhodamine isothiocyanate, rhodamine B dextran (D-1824) and lysinated tetramethylrhodamine dextran (fluoro-ruby, D-1817) have shown that the dextran conjugates produce less diffusion at injection sites and more permanent labeling than do the corresponding free dyes. 5 Dextran conjugates with molecular weights up to 70,000 daltons have been employed as neuronal tracers in a wide variety of species. The availability of fluorescent dextran conjugates with different sizes and charges permitted the analysis of direction and rate of axonal transport in the squid giant axon. 16 Multilabeled Dextrans Our fixable dextrans, most of which are lysinated dextrans (see the products marked by a single dagger ( ) in Table 14.4), are generally preferred for neuronal tracing because they may transport more effectively and can be fixed in place with aldehydes after labeling. Molecular Probes prepares a number of multilabeled dextrans that are fixable, including some that have acquired the distinction of unique names in various publications: Fluoro-ruby 5,13,17 22 a red-orange fluorescent, aldehydefixable 10,000 MW dextran labeled with both tetramethylrhodamine and lysine (D-1817; Figure 14.63) MW, 70,000 MW and 2,000,000 MW versions of fluoro-ruby are also available (D-3308, D-1818, D-7139). Fluoro-emerald 18,22,23 a green-fluorescent, aldehyde-fixable 10,000 MW dextran labeled with both fluorescein and lysine (D-1820; Figure 14.64, Figure 14.65, Figure 14.66) MW, 40,000 MW, 70,000 MW, 500,000 MW and 2,000,000 MW versions of fluoro-emerald are also available 24,25 (D-3306, D-1845, D-1822, D-7136, D-7137). Micro-ruby (D-7162) and mini-ruby (D-3312) red-orange fluorescent, aldehyde-fixable 3000 MW and 10,000 MW dextrans simultaneously labeled with tetramethylrhodamine, biotin and lysine. Micro-emerald (D-7156) and mini-emerald (D-7178) green-fluorescent, aldehyde-fixable dextrans simultaneously labeled with fluorescein, biotin and lysine. Section

4 Biotinylated dextran amine (BDA) 1,8,31 38 nonfluorescent, aldehyde-fixable dextrans simultaneously labeled with both biotin and lysine. These are available in several molecular weights (D-1956, D-1957, C-7135, D-7142; Table 14.4). A useful review has been published on the BDA products. 39 Figure Fluorescence excitation and emission spectra of tetramethylrhodamine-labeled 10,000 molecular weight dextran (fluoro-ruby, D-1817) in ph 7.0 buffer. Figure Lineage tracing of three dorsal blastomeres of 32-cell Xenopus laevis embryos injected with Molecular Probes fluorescent dextran conjugates. The tier 1 dorsal (A1) blastomere was injected with 10,000 MW lysine-fixable, fluorescein dextran (D-1820), the tier 2 dorsal (B1) blastomere with lysine-fixable Texas Red 10,000 MW dextran (D-1863), and the tier 3 dorsal (C3) blastomere with lysine-fixable Cascade Blue 10,000 MW dextran (D-1976). Embryos were fixed in formaldehyde, embedded and sectioned. The image on the left shows a 13 µm thick section of a stage 6 (32- cell) embryo fixed right after injection; this section exhibits significant autofluorescence due to the presence of residual yolk. The image on the right is a stage 10 (early gastrula) embryo. Triple-exposure photographs of the sectioned embryos were taken on a Zeiss Axiophot with a 10X objective. Images contributed by Marie Vodicka, Department of Molecular and Cell Biology, University of California, Berkeley. Our Influx pinocytic cell-loading reagent (I-14402, Section 20.8) is useful for loading dextran conjugates into some live cells. Fluoro-ruby and fluoro-emerald (Figure 14.66) have been extensively employed for retrograde and anterograde neuronal tracing, 5 transplantation 20 and cell-lineage tracing. 2,19,21,22,40 Both products can be used to photoconvert DAB into an insoluble, electrondense reaction product. 13 Like fluoro-ruby and fluoro-emerald, micro-ruby and mini-ruby are brightly fluorescent, making it easy to visualize the electrode during the injection process. DiI (D-282, Section 14.4) or other lipophilic probes in Section 14.4 can be used to mark the sites of microinjection. 41 In addition, because these dextrans include a covalently linked biotin, filled cells can be probed with standard enzyme-labeled avidin or streptavidin conjugates or with NANOGOLD and FluoroNanogold streptavidin (Section 7.6) to produce a permanent record of the experiment. 42 Mini-ruby has proven useful for intracellular filling in fixed brain slices 27 and has been reported to produce staining comparable to that achieved with lucifer yellow CH 26 (L-453, L-682, L-1177, L-12926; Section 14.3). Moreover, the use of mini-ruby in conjunction with standard peroxidasemediated avidin biotin methods does not cause co-conversion of lipofuscin granules found in adult human brain, a common problem during photoconversion of lucifer yellow CH. 26 The lysine-fixable micro-emerald and mini-emerald dextrans that are triply labeled with fluorescein, biotin and lysine provide a contrasting color that is better excited by the argon-ion laser of confocal laser-scanning microscopes; they have uses similar to micro-ruby and mini-ruby, respectively MW Dextrans The nominally 3000 MW dextrans offer several advantages over higher molecular weight dextrans, including faster axonal diffusion and greater access to peripheral cell processes 24 (Figure 14.67). Our 3000 MW dextran preparations contain polymers with molecular weight predominantly in the range of ~ daltons, including the dye or other label. Our list of 3000 MW dextrans includes fluorescein, Rhodamine Green, tetramethylrhodamine, Texas Red and biotin conjugates. We also offer lysine-fixable 3000 MW dextrans that are simultaneously labeled with both fluorescein and biotin (micro-emerald, D-7156) or tetramethylrhodamine and biotin (micro-ruby, D-7162). The 3000 MW fluorescein dextran and tetramethylrhodamine dextran (D-3306, D-3308; Figure 14.68, Figure 14.69) have been observed to readily undergo both anterograde and retrograde movement in live cells. 18,24 These 3000 MW dextrans appear to passively diffuse within the neuronal process, as their intracellular transport is not effectively inhibited by colchicine or nocodazole, both of which disrupt active transport by depolymerizing microtubules. 24 Moreover, these small dextrans diffuse at rates equivalent to those of smaller tracers such as sulforhodamine 101 and biocytin (~2 millimeters/hour at 22 C) and about twice as fast as 10,000 MW dextrans. The relatively low molecular weight of the dextrans may result in transport of some labeled probes through gap junctions (see below). By use of anti-tetramethylrhodamine antibodies (A-6397, Section 7.4) and peroxidase anti-peroxidase complex staining, the signal from tetramethylrhodamineconjugated dextrans can be detected in the fine dendrite configuration of cortical projection neurons. 12 NeuroTrace BDA-10,000 Neuronal Tracer Kit Designed for both the first-time user and the experienced neuroscientist, our Neuro- Trace BDA-10,000 Neuronal Tracer Kit (N-7167) contains convenient amounts of each of the components required for neuroanatomical tracing using BDA methods, 39 including: Lysine-fixable, biotinylated 10,000 MW dextran amine (BDA-10,000) Avidin horseradish peroxidase (avidin HRP) 3,3 -Diaminobenzidine (DAB) A rigorously tested protocol that ensures fast and simple tracing experiments The neuronal tracer BDA-10,000 is transported over long distances and fills fine processes bidirectionally, including boutons in the anterograde direction and dendritic 584 Chapter 14 Fluorescent Tracers of Cell Morphology and Fluid Flow

5 structures in the retrograde direction. 8,35 38 Two days to two weeks after BDA-10,000 is injected into the desired region of the brain, the brain tissue can be fixed and sectioned. BDA-10,000 can also be applied to cut nerves and allowed to transport. Following incubation with the avidin HRP conjugate and then DAB, the electron-dense DAB reaction product can be viewed by either light or electron microscopy 39,42 (Figure 14.70). The NeuroTrace BDA-10,000 labeling method can be readily combined with other anterograde or retrograde labeling methods or with immunohistochemical techniques. BDA- 10,000 is available as a separate product (D-1956), as are BDA derivatives with other molecular weights BDA (D-7135), BDA-70,000 (D-1957) and BDA-500,000 (D-7142). A detailed protocol that utilizes our BDA-10,000 probe, streptavidin HRP conjugate (S-911, Section 7.6) and tetramethylbenzidine to anterogradely label fine processes in neurons has been published. 1 Cell Lineage Tracing with Dextrans Fluorescent dextrans particularly the fluorescein and rhodamine conjugates have been used extensively for tracing cell lineage. 21,44,45 In this technique, the dextran is microinjected into a single cell of the developing embryo, and the fate of that cell and its daughters can be followed in vivo (Figure 14.64). Examples using this method include studies of: Dorsoventral axis determination in zebrafish mutants 46 Early cell-fate commitment and lineage restrictions in developing zebrafish Lineage and dopamine phenotype in tadpole hypothalamus 50 Migration of neural crest cells in Xenopus 51 Neural crest cell fate in zebrafish 52 Progeny tracing in the grasshopper neuroblast 8 Developmental studies show that the lysinated fluorescent dextrans are also suitable for cell ablation studies, presumably through the generation of oxygen radicals. 53 The lysine-fixable tetramethylrhodamine and Texas Red dextran conjugates (Table 14.4) are most frequently cited for lineage tracing studies; they are often preferred over other conjugates because they have bright fluorescence and are relatively photostable. Our Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594 and Alexa Fluor 647 dextrans (D-22911, D-22912, D-22913, D-22914) are likely to provide equal or superior performance as orange- to red-fluorescent polar tracers. As a second color, particularly in combination with the Texas Red dextrans, people have most often used our lysine-fixable fluorescein dextrans (e.g., D-3306, D-1820, D-1822). However, the photostability of fluorescein conjugates is not as high as that of the tetramethylrhodamine and Texas Red conjugates. Consequently, we recommend our green-fluorescent Alexa Fluor 488 dextran conjugate (D-22910) or, alternatively, our lysine-fixable Oregon Green 488 (D-7171, D-7173), Oregon Green 514 (D-7175) and Rhodamine Green (D-7153) dextran conjugates; see Figure 1.42, Figure 7.19 and Figure 11.8 for a comparison of the photostability of the Alexa Fluor 488, Oregon Green and Rhodamine Green dyes and fluorescein. The more photostable dextrans may also be less phototoxic in cells. Although these fixable conjugates can be employed with long-term preservation of the tissue, some researchers prefer to co-inject a fluorescent, nonlysinated dextran along with a nonfluorescent, lysine-fixable biotin dextran (BDA, Table 14.4). The nonfluorescent BDA can then be fixed in place with aldehyde-based fixatives and probed with any of our fluorescent or enzyme-labeled streptavidin and avidin conjugates described in Section 7.6 (Table 7.17). Vybrant Cell Lineage Tracing Kit The Vybrant Cell Lineage Tracing Kit (V-22915) combines an aldehyde-fixable Cascade Blue dextran with the superior brightness of our red-orange fluorescent Alexa Fluor 546 dye to permit the detection of widely differentiated cell lineages. The Cascade Blue dextran is first injected into the desired parent cells. After differentiation, the cells are fixed and the signal is amplified using a rabbit IgG antibody against the Cascade Blue dye and an Alexa Fluor 546 conjugate of goat anti rabbit IgG antibody as the detection reagent (Figure 7.65). The red-orange fluorescent Alexa Fluor 546 dye can be Figure Development of the leech central nervous system into segments was characterized with the cell lineage tracer, 10,000 MW lysine-fixable, fluorescein dextran (fluoro-emerald, D-1820). Fluoro-emerald was injected into a neuroectodermal cell of a leech embryo. The teloblast eventually differentiated into five segmentally iterated ganglia labeled with fluoro-emerald (pseudocolored blue in this image). Associated muscle fibers were identified with a Lan 3-14 monoclonal antibody and visualized with a Cy3 antibody (pseudocolored red). Nuclei were counterstained with TOTO-3 dye (T-3604, pseudocolored green). Image reprinted from the cover of Development 127 (4) (2000), used with the permission of The Company of Biologists Ltd. Figure A whole-mount of the embryonic brain of Xenopus laevis that has been doublelabeled with our 10,000 MW lysine-fixable, fluorescein dextran and tetramethylrhodamine dextran (fluoro-emerald, D-1820; fluoro-ruby, D-1817). The tetramethylrhodamine dextran was used to label the neurons projecting from the retina, whereas the fluorescein dextran was applied to the transected spinal cord, thus allowing the detailed evaluation of the topological relationship of these two populations of neurons (Neurosci Lett 127, 150 (1991)). Image contributed by Martina Manns and Bernd Fritzsch, Department of Biomedical Sciences, Creighton University. Section

6 viewed using filters appropriate for tetramethylrhodamine (Table 24.8). The Vybrant Cell Lineage Tracing Kit contains: Lysine-fixable, 10,000 MW, Cascade Blue dextran (3 vials) Rabbit IgG anti Cascade Blue antibody Alexa Fluor 546 goat anti rabbit IgG antibody conjugate A detailed experimental protocol Figure Secondary motor neurons in a spinal cord whole mount of a male western mosquitofish (Gambusia affinis affinis) that have been labeled with 3000 MW lysine-fixable, Texas Red dextran (D-3328). The dextran crystals were applied to the bipinnate inclinator muscles of the anal appendicular support fin, and the dye was transported from the axons to cell body and dendrites. Motor neurons were visualized and photographed through a bandpass optical filter appropriate for Texas Red dye, by epifluorescence microscopy. Image contributed by E. Rosa-Molinar, Department of Cell Biology and Anatomy, University of Nebraska Medical Center, and Bernd Fritzsch, Department of Biomedical Sciences, Creighton University. High MW Dextran Conjugates Our 500,000 and 2,000,000 MW fluorescent dextrans (Table 14.4) may be particularly useful for lineage tracing at early stages of development, although these biopolymers have lower water solubility and a greater tendency to precipitate or clog microinjection needles than our lower molecular weight dextrans. Some studies suggest that lower molecular weight dextrans may leak from blastomeres, complicating analysis. Injection of 2,000,000 MW fluorescein- and Texas Red dye conjugated dextrans into separate cells of the two-cell stage zebrafish embryo allowed the construction of a fate map. 54 The 500,000 MW and 2,000,000 MW dextrans are labeled with fluorescein, tetramethylrhodamine or Texas Red dyes or with biotin, and all contain aldehyde-fixable lysine groups. The nonfluorescent 500,000 MW aminodextran (D-7144) can be conjugated with the researcher s choice of amine-reactive reagents. Caged Fluorophore Dextrans Dextrans with caged fluorophores are of particular interest to developmental biologists, because they can be injected early in development when the cells are large, and then later activated with UV illumination when the cells of interest may be small or buried in tissue (Figure 17.7). A caged-fluorescein dextran conjugate has been used in this way to demonstrate lineage restriction boundaries in the early Drosophila embryo. 58 Two-photon excitation has been used to photoactivate a caged-fluorescein dextran at the two-cell stage of sea urchin embryo development. 59 A 10,000 MW dextran conjugate of DMNB-caged fluorescein (D-3310, Figure 17.7) is available (Table 14.4), as is a DMNB-caged fluorescein dextran (D-7146) that has been further modified with both lysine and biotin to make it fixable, as well as detectable by avidin conjugates. See Chapter 17 for a discussion of caged probes and Section 14.3 for a description of our lower molecular weight caged tracers for microinjection. Studying Intercellular Communication with Dextrans Figure The attached eighth nerve from the vestibular labyrinth of a turtle, Pseudemys scripta, exposed to 3000 MW lysine-fixable, tetramethylrhodamine dextran (D-3308) and incubated with F-actin specific BODIPY FL phallacidin (B-607). F-actin labeled ciliary bundles were stained green by the phallacidin, and the calyceal and bouton endings of their primary afferents were stained red by the dextran. This image is a projection of 40 confocal images. Image contributed by Laura Di- Caprio and Ellengene Peterson, Department of Biological Sciences, Ohio University. Caged fluorophore dextrans have particular utility for developmental biology studies. The fluorescence can be turned on with UV light at any stage of development and the fluorophore becomes a hapten for antibodies to the dye. The size of dextrans may be exploited to study connectivity between cells. Examples include studies of the passage of 3000 MW dextrans through plasmodesmata 25 and modulation of gap junctional communication by transforming growth factor β 1 and forskolin. 60 However, the dispersion of molecular weights in our 3000 MW dextran preparations, which contain polymers with total molecular weights predominantly in the range of ~ daltons but may also contain molecules <1500 daltons, may complicate such analyses. An important experimental approach to identifying cells that form gap junctions makes use of simultaneous introduction of the polar tracer lucifer yellow CH (~450 daltons) and a tetramethylrhodamine 10,000 MW dextran. Because low molecular weight tracers like lucifer yellow CH (L-453, L-12926; Section 14.3) pass through gap junctions and dextrans do not, the initially labeled cell exhibits red fluorescence, whereas cells connected through gap junctions have yellow fluorescence 60 (Figure 14.71) This technique has been used to follow the loss of intercellular communication in adenocarcinoma cells, 61 to show the re-establishment of communication during wound healing in Drosophila 62 and to investigate intercellular communication at different stages in Xenopus embryos. 63,64 Similar experiments could employ our lucifer yellow 10,000 MW dextran (D-1825) and a low molecular weight red-fluorescent tracer such as sulforhodamine 101 (S-359, Section 14.3) or a blue-fluorescent dye such as Cascade Blue hydrazide (C-687, Section 14.3). Simultaneous loading of cells with two (or more) dextrans that differ in both their molecular weight and in the dye s fluorescence properties has been used to assess subcellular heterogeneities in the submicroscopic structure of cytoplasm Chapter 14 Fluorescent Tracers of Cell Morphology and Fluid Flow

7 Probing Membrane Permeability with Dextrans Labeled dextrans are often used to investigate the exclusion or transfer of macromolecules across cell membranes. For example, fluorescent dextrans have been used to monitor the effectiveness of electroporation, a technique that produces pores in the cell membrane, thus providing a convenient method for introducing materials such as exogenous DNA. Fluorescein dextrans with molecular weights ranging from 4000 to 150,000 daltons were used to determine the effect of electroporation variables pulse size, shape and duration on plasma-membrane pore size in chloroplasts, 66 red blood cells 67 and fibroblasts. 68 Fluorescence recovery after photobleaching (FRAP) techniques have been used to monitor nucleocytoplasmic transport of fluorescent dextrans of various molecular weights, allowing the determination of the size-exclusion limit of the nuclear pore membrane, as well as to study the effect of epidermal growth factor and insulin on the nuclear membrane and on nucleocytoplasmic transport. 72,73 Microinjected 3000 MW fluorescent dextrans concentrate in interphase nuclei of Drosophila embryos, whereas 40,000 MW dextrans remain in the cytoplasm and enter the nucleus only after breakdown of the nuclear envelope during prophase. This size-exclusion phenomenon was used to follow the cyclical breakdown and reformation of the nuclear envelope during successive cell divisions. 74 Similarly, our 10,000 MW Calcium Green dextran conjugate (C-3713, Section 20.4) was shown to diffuse across the nuclear membrane of isolated nuclei from Xenopus laevis oocytes, but the 70,000 MW and 500,000 MW conjugates could not. 75 Significantly, depletion of nuclear Ca 2+ stores by inositol 1,4,5- triphosphate (Ins 1,4,5-P 3, I-3716; Section 18.2) or by calcium chelators (Section 20.8) blocked nuclear uptake of the 10,000 MW Calcium Green dextran conjugate but not entry of lucifer yellow CH. Our 3000 MW Calcium Green dextran conjugate (C-6765) is actively transported in adult nerve fibers over a significant distance and is retained in presynaptic terminals in a form that allows monitoring of presynaptic Ca 2+ levels. 76 Fluorescent dextrans with molecular weights up to 20,000 daltons are reported to be taken up by the feeding tubes of nematodes, but 40,000 MW and 70,000 MW dextrans are not. 77 Following Endocytosis and Fusion with Dextrans Some of the dyes that Molecular Probes uses to prepare its dextran conjugates exhibit fluorescence that is sensitive to the ph of the medium (Chapter 21). Consequently, internalization of labeled dextrans into acidic organelles of cells can often be tracked by measuring changes in the fluorescence of the dye. 78,79 Fluorescence of fluorescein-labeled dextrans is strongly quenched upon acidification (Figure 21.2); however, fluorescein s lack of a spectral shift in acidic solution makes it difficult to discriminate between internalized probe that is quenched and the residual fluorescence of the external medium. Dextran conjugates that either shift their emission spectra, such as the SNARF and SNAFL dextrans, or undergo significant shifts of their excitation spectra, such as BCECF, Oregon Green and HPTS dextrans, are much more useful for following the internalization by ratio imaging (see Loading and Calibration of Intracellular Ion Indicators in Section 20.1). Our ph indicator conjugates and their optical responses are described in Section Discrimination of internalized fluorescent dextrans from external dextrans can be improved by adding a reagent that quenches the fluorescence of the external probe. For example, our anti-dye antibodies (Section 7.4) usually quench the fluorescence of their cognate dyes and may be useful for discriminating between externally bound dextrans and internalized dextrans. In addition, trypan blue can also be used as a quencher for some of the external dextran conjugates. Fluorescent dextrans can also be encapsulated in liposomes Using Texas Red and fluorescein-labeled dextrans encapsulated in liposomes, researchers have obtained evidence that antigen processing occurs within dense lysosomes, rather than in earlier endocytic compartments. 83 Researchers have also used liposome-encapsulated fluorescent dextrans to investigate liposome fusion with isolated nuclei 84 and the effect of additives on vesicle size. 85 Intracellular fusion of endosomes has been followed by using the fluorescence enhancement of BODIPY FL avidin that occurs when it complexes with a biotinylated dextran 86 (Figure 16.24). We have found our Oregon Green 514 streptavidin (S-6369, Section 7.6) to have an over 15-fold increase in fluorescence intensity upon Figure The antero-ventral and antero-dorsal lateral line nerves in an Ambystoma mexicanum whole-mounted brain after labeling with 3000 MW Rhodamine Green dextran (D-7163) and 3000 MW lysine-fixable, tetramethylrhodamine dextran (D-3308), respectively. In both cases, the dextran was applied to the respective cut cranial nerves. This photomicrograph shows the four segregated ventral fascicles of the mechanosensory lateral line fibers and the intermingling of the dorsal electrosensory fibers. Image contributed by Bernd Fritzsch, Department of Biomedical Sciences, Creighton University. Figure A mitral cell in the olfactory bulb of a chinook salmon that has been retrogradely labeled using our NeuroTrace BDA-10,000 Neuronal Tracer Kit (N-7167). The combination of low molecular weight fluorescent dyes and dextrans is extensively utilized to study gap junction formation. Section

8 binding free biotin, which may make it the preferred probe for this application (Figure 16.24). Tracing Fluid Transport with Dextrans Fluorescent dextrans are important tools for studying the hydrodynamic properties of the cytoplasmic matrix. The intracellular mobility of these fluorescent tracers can be investigated using fluorescence recovery after photobleaching (FRAP) techniques. We offer a range of dextran sizes, thus providing a variety of hydrodynamic radii for investigating both the nature of the cytoplasmic matrix and the permeability of the surrounding membrane. Because of their solubility and biocompatibility, fluorescent dextrans have been used to monitor in vivo tissue permeability and flow in the uveoscleral tract, 87,88 capillaries 89,90 and proximal tubules, 91 as well as diffusion of high molecular weight substances in the brain s extracellular environment. 92 Fluorescent dextrans have also been used to assess permeability of the blood brain barrier 93 and to monitor blood flow. 94 Our 10,000 MW DMNB-caged fluorescein dextran (D-3310, Figure 17.7) and the corresponding triple-labeled DMNB-caged fluorescein, biotin and lysine dextran (D-7146) are fluorescent only after UV photolysis, enabling researchers to conduct photoactivation of fluorescence (PAF) experiments analogous to FRAP experiments in which the fluorophore is photoactivated A upon illumination rather than bleached (Figure 7.93). Measuring the bright signal of the photoactivated fluorophore against a dark background should be intrinsically more sensitive than measuring a dark (photobleached) region against a bright field. In a collaboration with Walter Lempert of Princeton University, we have shown caged fluorescein dextran (D-3310) to be an effective probe for tracing vortices in water using a technique called photoactivated nonintrusive tracking of molecular motion (PHAN- TOMM). 99 Furthermore, diffusional coupling between dendritic spines and shafts was measured both by FRAP experiments with fluorescein dextran and by PAF experiments with DMNB-caged fluorescein dextran. 100 DMNB-caged fluorescein was also employed to evaluate a system that combined confocal laser-scanning microscopy with local photolysis of caged compounds. 95 Our Bibliography of Dextran Applications Our bibliography of dextran applications contains over 1100 references. It includes references in which dextrans from several different sources were used. Because the source, molecular weight of the dextran, net charge, degree of substitution and nature of the dye may significantly affect the application, the methods described in this section and in the references in our bibliography should be considered guides rather than definitive protocols. In most cases, however, our fluorescent dextrans are much brighter and have higher negative charge than dextrans available from other sources. Furthermore, we use rigorous methods for removing as much unconjugated dye as practical, and then assay our dextran conjugates by thin-layer chromatography to ensure the absence of low molecular weight contaminants. References B Figure Dual tracer technique for identification of gap junction coupled cells. A) Cells are labeled with a mixture of a small polar tracer such as lucifer yellow CH (green circles) and a relatively large tetramethylrhodamine-labeled dextran (red circles). B) Adjoining gap junction coupled cells are accessible to the low molecular weight tracer whereas the much larger dextran conjugate is excluded. Coupled cells with single-color lucifer yellow CH labeling are readily distinguished from initially labeled cells with dual fluorescence. 1. J Neurosci Methods 109, 81 (2001); 2. Brain Res Bull 51, 11 (2000); 3. J Cell Sci 96, 721 (1990); 4. J Cell Biol 105, 1981 (1987); 5. Brain Res 526, 127 (1990); 6. Brain Res Bull 25, 139 (1990); 7. J Neurosci Methods 81, 169 (1998); 8. J Neurosci 14, 5766 (1994); 9. Plant Physiol 98, 673 (1992); 10. Plant Physiol 93, 1492 (1990); 11. Biotech Histochem 67, 153 (1992); 12. J Neurosci Methods 65, 157 (1996); 13. J Histochem Cytochem 41, 777 (1993); 14. Brain Res Bull 30, 115 (1993); 15. Trends Neurosci 13, 14 (1990); 16. Proc Natl Acad Sci U S A 92, (1995); 17. Mol Biol Cell 6, 1491 (1995); 18. J Neurosci Methods 53, 35 (1994); 19. Nature 363, 630 (1993); 20. Proc Natl Acad Sci U S A 90, 1310 (1993); 21. Nature 344, 431 (1990); 22. Dev Biol 109, 509 (1985); 23. J Cell Biol 128, 293 (1995); 24. J Neurosci Methods 50, 95 (1993); 25. Plant J 4, 567 (1993); 26. J Neurosci Methods 55, 105 (1994); 27. Brain Res 608, 78 (1993); 28. J Neurosci Methods 74, 9 (1997); 29. Glia 20, 145 (1997); 30. Hippocampus 8, 57 (1998); 31. J Neurosci 15, 5222 (1995); 32. J Neurosci 15, 5139 (1995); 33. J Neurosci Methods 53, 23 (1994); 34. J Neurosci Methods 52, 153 (1994); 35. Brain Res 607, 47 (1993); 36. J Neurosci Methods 48, 75 (1993); 37. J Neurosci Methods 45, 35 (1992); 38. J Neurosci Methods 41, 239 (1992); 39. J Neurosci Methods 103, 23 (2000); 40. Development 104 Suppl, 231 (1988); 41. Biophys J 75, 2558 (1998); 42. Neuroscience Protocols, Wouterlood FG, Ed , pp (1993); 43. J Neurosci Methods 76, 167 (1997); 44. Science 252, 569 (1991); 45. Development 108, 581 (1990); 46. Nature 370, 468 (1994); 47. Development 120, 483 (1994); 48. Science 265, 517 (1994); 49. Science 261, 109 (1993); 50. J Neurosci 12, 1351 (1992); 51. Development 118, 363 (1993); 52. Development 120, 495 (1994); 53. Dev Biol 120, 520 (1987); 54. Nature 361, 451 (1993); 55. Dev Biol 201, 247 (1998); 56. Methods Mol Biol 135, 349 (2000); 57. Biochem Cell Biol 75, 551 (1997); 58. Cell 68, 923 (1992); 59. Dev Biol 175, 177 (1996); 60. J Neurosci 15, 262 (1995); 61. Proc Natl Acad Sci U S A 85, 473 (1988); 62. Dev Biol 127, 197 (1988); 63. J Cell Biol 110, 115 (1990); 64. Dev Biol 129, 265 (1988); 65. J Immunol 157, Chapter 14 Fluorescent Tracers of Cell Morphology and Fluid Flow

9 References continued (1996); 66. Biophys J 58, 823 (1990); 67. Bioelectrochem Bioenerg 20, 57 (1988); 68. Biotechniques 6, 550 (1988); 69. J Cell Biol 102, 1183 (1986); 70. EMBO J 3, 1831 (1984); 71. J Biol Chem 258, (1983); 72. J Cell Biol 110, 559 (1990); 73. Biochemistry 26, 1546 (1987); 74. Biotechniques 17, 730 (1994); 75. Science 270, 1835 (1995); 76. Neurosci Lett 258, 124 (1998); 77. Parasitology 109, 249 (1994); 78. FASEB J 8, 573 (1994); 79. J Cell Sci 105, 861 (1993); 80. Agr Biol Chem 50, 399 (1986); 81. FEBS Lett 179, 148 (1985); 82. J Cell Biol 99, 1989 (1984); 83. Cell 64, 393 (1991); 84. Biochemistry 26, 765 (1987); 85. Biochemistry 29, 4582 (1990); 86. Biophys J 71, 487 (1996); 87. Proc Natl Acad Sci U S A 85, 2315 (1988); 88. Arch Ophthalmol 105, 844 (1987); 89. Microvasc Res 36, 172 (1988); 90. Am J Physiol 245, H495 (1983); 91. Am J Physiol 253, F366 (1987); 92. Biophys J 65, 2277 (1993); 93. Pflugers Arch 427, 86 (1994); 94. J Cereb Blood Flow Metab 13, 359 (1993); 95. Neuron 15, 755 (1995); 96. Anal Chem 70, 2459 (1998); 97. Biophys J 74, 3302 (1998); 98. AIAA Journal 34, 449 (1996); 99. Exp Fluids 18, 249 (1995); 100. Science 272, 716 (1996). Product List 14.5 Fluorescent and Biotinylated Dextrans Cat # Product Name Unit Size D dextran, Alexa Fluor 488; 10,000 MW, anionic, fixable... 5 mg D dextran, Alexa Fluor 546; 10,000 MW, anionic, fixable... 5 mg D dextran, Alexa Fluor 568; 10,000 MW, anionic, fixable... 5 mg D dextran, Alexa Fluor 594; 10,000 MW, anionic, fixable... 5 mg D dextran, Alexa Fluor 647; 10,000 MW, anionic, fixable... 2 mg D-3330 dextran, amino, 3000 MW mg D-1860 dextran, amino, 10,000 MW... 1 g D-1861 dextran, amino, 40,000 MW... 1 g D-1862 dextran, amino, 70,000 MW... 1 g D-7144 dextran, amino, 500,000 MW mg D-7135 dextran, biotin, 3000 MW, lysine fixable (BDA-3000) mg D-1956 dextran, biotin, 10,000 MW, lysine fixable (BDA-10,000) mg D-1957 dextran, biotin, 70,000 MW, lysine fixable (BDA-70,000) mg D-7142 dextran, biotin, 500,000 MW, lysine fixable (BDA-500,000) mg D-7168 dextran, BODIPY FL, 10,000 MW, fixable... 5 mg D-7132 dextran, Cascade Blue, 3000 MW, anionic, lysine fixable mg D-1976 dextran, Cascade Blue, 10,000 MW, anionic, lysine fixable mg D-3310 dextran, DMNB-caged fluorescein, 10,000 MW, anionic... 5 mg D-7146 dextran, DMNB-caged fluorescein and biotin, 10,000 MW, lysine fixable... 5 mg D-3305 dextran, fluorescein, 3000 MW, anionic mg D-3306 dextran, fluorescein, 3000 MW, anionic, lysine fixable mg D-1821 dextran, fluorescein, 10,000 MW, anionic mg D-1820 dextran, fluorescein, 10,000 MW, anionic, lysine fixable (fluoro-emerald) mg D-1844 dextran, fluorescein, 40,000 MW, anionic mg D-1845 dextran, fluorescein, 40,000 MW, anionic, lysine fixable mg D-1823 dextran, fluorescein, 70,000 MW, anionic mg D-1822 dextran, fluorescein, 70,000 MW, anionic, lysine fixable mg D-7136 dextran, fluorescein, 500,000 MW, anionic, lysine fixable mg D-7137 dextran, fluorescein, 2,000,000 MW, anionic, lysine fixable mg D-7156 dextran, fluorescein and biotin, 3000 MW, anionic, lysine fixable (micro-emerald)... 5 mg D-7178 dextran, fluorescein and biotin, 10,000 MW, anionic, lysine fixable (mini-emerald) mg D-1825 dextran, lucifer yellow, 10,000 MW, anionic, lysine fixable mg D-7170 dextran, Oregon Green 488; 10,000 MW, anionic... 5 mg D-7171 dextran, Oregon Green 488; 10,000 MW, anionic, lysine fixable... 5 mg D-7172 dextran, Oregon Green 488; 70,000 MW, anionic... 5 mg D-7173 dextran, Oregon Green 488; 70,000 MW, anionic, lysine fixable... 5 mg D-7175 dextran, Oregon Green 514; 10,000 MW, anionic, lysine fixable... 5 mg D-7176 dextran, Oregon Green 514; 70,000 MW, anionic... 5 mg D-1824 dextran, rhodamine B, 10,000 MW, neutral mg D-1841 dextran, rhodamine B, 70,000 MW, neutral mg D-7163 dextran, Rhodamine Green, 3000 MW... 5 mg D-7153 dextran, Rhodamine Green, 10,000 MW, lysine fixable mg D-3307 dextran, tetramethylrhodamine, 3000 MW, anionic mg D-3308 dextran, tetramethylrhodamine, 3000 MW, anionic, lysine fixable mg D-1868 dextran, tetramethylrhodamine, 10,000 MW, anionic, fixable mg D-1817 dextran, tetramethylrhodamine, 10,000 MW, lysine fixable (fluoro-ruby) mg D-1816 dextran, tetramethylrhodamine, 10,000 MW, neutral mg D-1842 dextran, tetramethylrhodamine, 40,000 MW, neutral mg D-1818 dextran, tetramethylrhodamine, 70,000 MW, lysine fixable mg D-1819 dextran, tetramethylrhodamine, 70,000 MW, neutral mg D-7139 dextran, tetramethylrhodamine, 2,000,000 MW, lysine fixable mg D-7162 dextran, tetramethylrhodamine and biotin, 3000 MW, lysine fixable (micro-ruby)... 5 mg Section

10 Product List 14.5 Fluorescent and Biotinylated Dextrans continued Cat # Product Name Unit Size D-3312 dextran, tetramethylrhodamine and biotin, 10,000 MW, lysine fixable (mini-ruby) mg D-3328 dextran, Texas Red, 3000 MW, lysine fixable mg D-3329 dextran, Texas Red, 3000 MW, neutral mg D-1863 dextran, Texas Red, 10,000 MW, lysine fixable mg D-1828 dextran, Texas Red, 10,000 MW, neutral mg D-1829 dextran, Texas Red, 40,000 MW, neutral mg D-1864 dextran, Texas Red, 70,000 MW, lysine fixable mg D-1830 dextran, Texas Red, 70,000 MW, neutral mg N-7167 NeuroTrace BDA-10,000 Neuronal Tracer Kit... 1 kit V Vybrant Cell Lineage Tracing Kit... 1 kit 14.6 FluoSpheres and TransFluoSpheres Microspheres for Tracing Figure Normalized fluorescence emission spectra of the dyes contained in FluoSpheres polystyrene microspheres for blood flow determination, after extraction into 2-ethoxyethyl acetate (Cellosolve acetate). The eleven colors of fluorescent microspheres represented are: 1) blue, 2) blue-green, 3) green, 4) yellow-green, 5) yellow, 6) orange, 7) red-orange, 8) red, 9) carmine, 10) crimson and 11) scarlet (Table 14.5). Molecular Probes Microsphere Products and Prices The catalog numbers and prices of all of our FluoSpheres microsphere products for tracing are listed in the tables of this section. Our complete line of luminescent microspheres is discussed in Section 6.5. FluoSpheres and TransFluoSpheres polystyrene microspheres 1 satisfy several prerequisites of ideal long-term biological tracers. Because the dyes in our microspheres are incorporated throughout the microsphere rather than just on its surface, the fluorescence output per microsphere is significantly greater than that obtained from protein or dextran conjugates (Table 6.6) and is relatively immune to photobleaching and other environment-dependent effects. FluoSpheres and TransFluoSpheres microspheres are also biologically inert and physically durable, and they are available from Molecular Probes with a large number of uniform sizes and surface properties. Furthermore, their spectral properties can be freely manipulated during manufacture without altering their surface properties. Our current carboxylate-modified fluorescent microspheres have much higher surface charges than were previously available, making them more hydrophilic and thus more useful as tracers. The additional carboxylate groups also make it easier to couple the microspheres to proteins and other biomolecules. See Section 6.5 for an extensive discussion of the properties of our FluoSpheres and TransFluoSpheres polystyrene beads. Molecular Probes also makes numerous fluorescent microsphere products that are useful as standards for imaging (Section 24.1) and flow cytometry (Section 24.2). Availability of intensely fluorescent, highly uniform microspheres in different colors and sizes permits diverse applications in tracking particles and cells, tracing fluid dynamics and amplifying signals. In addition, measuring the effect of various interventions on regional blood flow is an important quantitative application of fluorescent microspheres. Using a mixture of beads of different sizes, each labeled with a different fluorescent color, researchers can discriminate the size dependence of uptake or transport of microspheres in vivo in cells, capillaries, lung or other tissues. 2 Our smallest microspheres can be microinjected into cells (see below) or are actively taken up by phagocytosis (Section 16.1). Fluorescent Microspheres for Regional Blood Flow Studies Relatively large radiolabeled microspheres (10 15 µm in diameter) have long been used for regional blood flow studies in tissues and organs. However, fluorescent microspheres have been shown to be superior to radioactive microspheres in chronic blood flow measurements. 3 In most cases, the microspheres are injected at desired locations in the circulatory system and eventually lodge in the capillaries, where they can later be counted in dissected tissue sections. To eliminate the hazards, expense and disposal problems of the radiolabeled microspheres, 4 researchers have turned to fluorescent and colored microspheres for measuring myocardial and cortical blood flow In addition, blood flow in brain cortex has been measured using much smaller microspheres (1.3 µm) by determining velocities of individual fluorescent beads with strobe epi-illumination. 12 Blood flow measurements using fluorescent microspheres in other organs, including the 590 Chapter 14 Fluorescent Tracers of Cell Morphology and Fluid Flow