Cell membranes provide a convenient conduit for loading live and fixed cells with lipophilic dyes. Not only can cells tolerate a high concentration of the lipophilic dye, but also lateral diffusion of the dye within the membrane can serve to stain the entire cell, even if the dye is applied locally. These properties have made lipophilic carbocyanine and aminostyryl dyes particularly important for anterograde and retrograde tracing in neuronal cells. Lipophilic tracers are used to label cells, organelles, liposomes, viruses and lipoproteins in a wide variety of long-term tracing applications, including cell transplantation, migration, adhesion and fusion studies. The distinguishing features of these carbocyanine and aminostyryl tracers are summarized in Summary of Molecular Probes lipophilic carbocyanine and aminostyryl tracers—Table 14.3. Other lipophilic probes described in Probes for Lipids and Membranes—Chapter 13 have also been used as tracers for liposomes. For example, incorporation of a BODIPY FL cholesterol derivative (C3927MP, Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3) in liposomes is virtually irreversible and the dye-labeled liposomes have been conjugated to antibodies for immunotargeting applications.
DiI, DiO, DiD and DiR
The lipophilic carbocyanines DiI (DiIC18(3), ), DiO (DiOC18(3), ), DiD (DiIC18(5), ) and DiR (DiIC18(7), ) are weakly fluorescent in water but highly fluorescent and quite photostable when incorporated into membranes (). They have extremely high extinction coefficients (EC >125,000 cm-1M-1 at their longest-wavelength absorption maximum) though modest quantum yields, and short excited-state lifetimes (~1 nanosecond) in lipid environments. Once applied to cells, the dyes diffuse laterally within the plasma membrane, resulting in staining of the entire cell. Transfer of these probes between intact membranes is usually but not always negligible. DiI and its analogs usually exhibit very low cell toxicity; however, moderate inhibition of electron transport chain activity has been reported for some analogs.
DiI, DiO, DiD and DiR exhibit distinct orange, green, red and infrared fluorescence, respectively (Figure 14.4.1), thus facilitating multicolor imaging. DiO (D275) and DiI (D282, D3911) can be used with standard fluorescein and rhodamine optical filters, respectively. Iontophoretic application of these lipophilic dyes to nerve terminals at a single neuromuscular junction in live animals permits tracing of other synaptic terminals of the same motor unit. The He-Ne laser–excitable DiD (D307, D7757) has much longer-wavelength excitation and emission spectra than those of DiI, providing a valuable alternative for labeling cells and tissues that have significant intrinsic fluorescence at wavelengths in the same range as DiI fluorescence (). DiD was used as a population marker in a flow cytometry study that also employed indo-1 to monitor intracellular Ca2+ mobilization. Our heptamethine carbocyanine probe DiIC18(7) (DiR, D12731) has even further red-shifted spectra, with absorption and emission maxima in the near-infrared region (absorption/emission maxima = 748/780 nm in methanol, Figure 14.4.1). Because the fluorescence of this dye is invisible to the human eye, it must be detected using a CCD camera or other near-infrared photosensitive device. The high transmission of infrared light through cells and tissues and low level of autofluorescence in the infrared makes DiR particularly useful as an in vivo tracer for labeled cells and liposomes in live organisms. Photoconversion of diaminobenzidine (DAB) by lipid tracers, including DiI and DiO derivatives, produces an insoluble, electron-dense reaction product (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2).
Figure 14.4.1 Normalized fluorescence emission spectra of DiO (D282), DiI (D282), DiD (D282) and DiR (D282) bound to phospholipid bilayer membranes.
Vybrant DiI, DiO and DiD Cell-Labeling Solutions
The highly lipophilic nature of DiI, DiO and DiD has often posed an obstacle to uniform cellular labeling in aqueous culture media. This technical difficulty has somewhat limited the use of these tracers in cell–cell fusion, cellular adhesion and migration applications. The Vybrant DiI cell-labeling solution (V22885) is a dye-delivery solution that can be added directly to normal culture media to uniformly label suspended or attached culture cells. The complementary Vybrant DiO and DiD cell-labeling solutions (V22886, V22887) allow cell populations to be marked in distinctive fluorescent colors for identification after mixing (Figure 14.4.1). Cells that have fused or that have formed stable clusters can be identified by double labeling (Figure 14.4.2). Each 1 mM solution of DiI, DiO or DiD has been filtered through a 0.2 µm polycarbonate filter. All three cell-labeling solutions are also available in the Vybrant Multicolor Cell-Labeling Kit (V22889). A Vybrant CM-DiI labeling solution is also available (V22888, see below under CM-DiI).
Figure 14.4.2 Poly(ethylene glycol)–induced fusion of Jurkat cells detected by flow cytometry. Two populations of Jurkat cells were separately labeled, one with the Vybrant DiI cell-labeling solution (V22885) and the other with the Vybrant DiO cell-labeling solution (V22886). Equal portions (1 mL) of the labeled cell suspensions were combined and treated with poly(ethylene glycol) for 45 seconds to induce fusion. The mixed-cell population was analyzed by flow cytometry. Double-labeled fused cells appear in the upper right quadrant of this bivariate correlation plot.
NeuroTrace DiI, DiO and DiD Tissue-Labeling Pastes
NeuroTrace DiI and NeuroTrace DiO tissue-labeling pastes (N22880, N22881) consist of DiI and DiO, respectively, mixed into an inert, water-resistant gel. Three formulations—NeuroTrace DiI, NeuroTrace DiO and NeuroTrace DiD—are available in the convenient NeuroTrace Multicolor Tissue-Labeling Kit (N22884). A NeuroTrace CM-DiI tissue-labeling paste is also available (N22883, see below under CM-DiI). These pastes are ready to use as supplied and can be applied directly to live or fixed tissue specimens using the tip of a needle. This method of application improves the penetration of the dye into bundled neurons, labeling axons both on and below the surface. In similar situations, direct application of dye crystals or microinjection of concentrated solutions will only label neurons on the surface. This labeling method has also been found to increase the rate of dye transport by 50–80% (H. Richard Koerber, University of Pittsburgh School of Medicine, personal communication).
FAST DiI and FAST DiO
Diffusion of lipophilic carbocyanine tracers from the point of their application to the terminus of a neuron can take several days to weeks. The diffusion process appears to be accelerated by introducing unsaturation in the alkyl tails of the probes. FAST DiI (D3899, D7756; ) and FAST DiO (D3898, ) have diunsaturated linoleyl (C18:2) tails in place of the saturated octadecyl tails (C18:0) of DiI and DiO. Migration of the unsaturated analogs is reported to be at least 50% faster than that of DiI and DiO (Andrea Elberger, University of Tennessee, personal communication). FAST DiI and the shorter chain DiIC12(3) (D383) internalize during endocytic sorting of lipids and are mainly found in the endocytic recycling compartment, whereas the 16-carbon DiIC16(3) (D384) is delivered to late endosomes. The perchlorate salt of FAST DiI does not crystallize and is a viscous oil (D3899); however, the 4-chlorobenzenesulfonate salt of FAST DiI (D7756) is a solid, making its crystals suitable for direct application to cells. We also offer a monounsaturated cis-9-octadecenyl (C18:1) analog of DiI (Δ9-DiI, D3886).
CellTracker CM-DiI (C7000, C7001) is a DiI derivative that is somewhat more water-soluble than DiIC18(3), thus facilitating the preparation of staining solutions for cell suspensions and fixed cells. In addition to its improved solubility in culture medium, CellTracker CM-DiI contains a thiol-reactive chloromethyl moiety () that allows the dye to covalently bind to cellular thiols. Thus, unlike other membrane stains, the label is well retained in some cells throughout fixation and permeabilization steps (Figure 14.4.3, ). Membrane staining with CellTracker CM-DiI persists following routine paraffin processing. CellTracker CM-DiI is particularly useful in experiments that combine membrane labeling with subsequent immunohistochemical analysis, fluorescence in situ hybridization or electron microscopy (). CM-DiI labeling of mesenchymal stem cells has been used to track their distribution in vivo and differentiation following transplantation. Similarly, cationic liposome complexes of DNA have been labeled with CM-DiI to identify their organ and cellular distribution following injection into mice.
In addition to providing CM-DiI as a 1 mg solid (C7001) or specially packaged as a set of 20 vials, each containing 50 µg of CM-DiI (C7000), we have available the Vybrant CM-DiI cell-labeling solution (V22888) and NeuroTrace CM-DiI tissue-labeling paste (N22883) for the most efficient labeling of cell suspension and tissues, respectively, with this useful aldehyde-fixable tracer.
Figure 14.4.3 Persistence of lipophilic tracer fluorescence following fixation. Cultured human B cells were stained with 20 µM SP-DiOC18(3) (D7778), SP-DiIC18(3) (D7777), CM-DiI (C7000, C7001) or PKH26 and then fixed with 3.7% formaldehyde or 3.7% formaldehyde + acetone. The fixed cells were analyzed by flow cytometry to generate a comparison of their fluorescence with that of the original live-cell population.
Because of poor solubility in water, it is often difficult to load the long-chain carbocyanine dyes such as DiI, DiO and PKH dyes into cells in suspension. To facilitate the staining of cells with long-chain carbocyanine dyes, we have developed sulfonated cyanine dyes —SP-DiIC18(3) (D7777, ), SP-DiOC18(3) (D7778), DiIC18(3)-DS (D7776, ) and DiIC18(5)-DS (D12730)—that retain the 18-carbon lipophilic chains of DiI, DiD and DiO but exhibit improved solubility in culture medium. Cells can be labeled with these dyes by simply diluting a DMSO stock solution of the dye to a labeling concentration of 1–10 µM in unmodified culture medium. We have determined that these sulfonated dyes are completely soluble at 20 µM for at least one hour in Dulbecco's phosphate-buffered saline and Hanks' balanced salt solution; aggregation begins after a few hours in solution. Uptake of the anionic carbocyanine dyes in live cells is usually slower than that of cationic carbocyanine dyes, and the resulting staining patterns may be quite different.
Some applications require retention of the membrane stain in place following aldehyde-mediated fixation and lipid extraction. In contrast to labeling with DiI, DiO or the PKH dyes, cell labeling with some of these sulfonated carbocyanines appears to be compatible with standard aldehyde-based fixation methods and acetone treatment, at least in some cell lines. Although the mechanism for cell retention of the sulfonated carbocyanines has not been determined, we have observed excellent retention of staining of human B cells with SP-DiIC18(3) and SP-DiOC18(3), whereas virtually all of the PKH26 dye (which is structurally identical to DiIC18(3)) is lost during the lipid extraction step (Figure 14.4.3). CM-DiI (C7000, C7001, N22883, V22888; see above) is also well retained during this processing. Furthermore, we have observed that acetone treatment actually enhances the fluorescence of some cells stained with SP-DiIC18(3) and especially with SP-DiOC18(3), a phenomenon that has not been seen with other carbocyanine-derived membrane stains.
Other DiO and DiI Analogs
Moderately lipophilic carbocyanines are also useful tracers for labeling cell membranes. Some scientists find the slightly less lipophilic DiIC12(3) (D383, ), DiIC16(3) (D384) and DiOC16(3) (D1125) easier to load into cell suspensions than their C18 homologs.
Neuronal Tracing Studies with DiI and DiO Analogs
DiI, DiO and DiD are widely used to label neuronal projections in live and fixed tissue (Figure 14.4.4, , ). The dyes insert into the outer leaflet of the plasma membrane and diffuse laterally, producing detailed labeling of fine neuronal projections. DiI has been reported to diffuse about 6 mm/day in live tissue but more slowly in fixed specimens. The dyes usually do not transfer from labeled to unlabeled cells, unless the membrane of the labeled cell is disrupted, and apparently do not transfer through gap junctions.
Motoneurons labeled with DiI have been reported to remain viable up to four weeks in culture and one year in vivo. Staining of neurons in fixed tissue with DiI has been reported to persist for at least two years. DiI and DiD can be used simultaneously for two-color tracing () and as a fluorescence donor–acceptor pair in excited-state energy transfer studies. Combinations of DiO, DiI, DiD and DiR may permit three- or even four-color measurements (Figure 14.4.1).
Cell labeling with the lipophilic carbocyanines is generally performed by direct application of a dye crystal or dye-coated paper, metal or glass probe or by microinjection onto single cells from a solution in dimethylformamide or dimethylformamide/ethanol. A method has been described for iontophoretic loading of cells with DiI and other lipid tracers. We also offer DiI in the form of extra-large crystals (D3911), which many researchers prefer for direct application to tissue. Detailed protocols for DiI labeling and confocal laser-scanning microscopy have been published. DiI has also been used to mark sites of microinjection by coating the outside of the glass micropipette with the dye. "DiOlistic labeling" of live or fixed cells and tissues uses a gene gun to deliver pellets coated with lipophilic carbocyanine dyes DiO, DiI and DiD, permitting rapid labeling at high densities with relatively high spatial resolution.
Figure 14.4.4 Visualizing gustatory neurons in a mouse tongue using DiI (D282, D3911). This wild-type mouse embryo was fixed in 4% phosphate-buffered paraformaldehyde, and taste ganglia were labeled with the lipophilic tracer DiI. Images were taken with an Olympus confocal microscope. Image provided by Robin F. Krimm, Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine.
Long-Term Cell Tracing with Membrane Tracers
Lipophilic carbocyanine tracers like DiI are also ideal labels for long-term cell tracing in vivo and in vitro, including studies of cell migration, transplantation, adhesion and fusion. The presence of DiI and DiO in the cell membrane does not appreciably affect cell viability, development or other basic physiological properties. Labeling of cell suspensions or perfusion of tissues requires aqueous dispersions of the dyes. Stock solutions of most of the tracers may be prepared in alcohol or dimethylsulfoxide (DMSO) and then added to cell suspensions. Several analogs of DiI that are more soluble in culture medium should facilitate this method of labeling (Summary of Molecular Probes lipophilic carbocyanine and aminostyryl tracers—Table 14.3).
Tissue Processing and Electron Microscopy with Long-Chain Carbocyanines
Methods have been developed to allow immunofluorescent labeling of tissue containing neurons stained with DiI. Polyacrylamide has been employed to embed DiI-stained brain tissue for vibratome sectioning —a method that has been reported to preserve DiI labeling better than cryostat sectioning. Also, see Summary of Molecular Probes lipophilic carbocyanine and aminostyryl tracers—Table 14.3 for a description of DiI and DiO analogs that may be better retained through fixation and permeabilization procedures.
Reports from several researchers indicate that DiA (4-Di-16-ASP, D3883, ) is a better neuronal tracer than DiO for multicolor labeling with DiI. DiA diffuses more rapidly in membranes and is more soluble than DiO, thus facilitating cell labeling. For example, DiA and DiI have been used together to investigate the interactions between dorsal root axons and their targets and axon outgrowth in the retina. DiA can be excited between 440 nm and 500 nm, and its maximum emission in DOPC vesicles is at ~590 nm (red-orange fluorescence, ). Its fluorescence in cells, however, is usually bright green to yellow-green (depending on the optical filter set used). The polyunsaturated FAST DiA, offered as a crystalline solid (D7758) form, may diffuse faster in membranes than DiA. The C10 analog of DiA, 4-Di-10-ASP (D291), has been used as a retrograde tracer to monitor injury-induced degradation of rat neurons in vivo and the role of microglial cells in removing debris resulting from natural and axotomy-induced neuronal cell death (). 4-Di-10-ASP staining of microglia can persist for at least 12 months and can be used for DAB photoconversion (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2). Axonal outgrowth of retinal ganglion cells stained with 4-Di-10-ASP has been observed.
For researchers wishing to investigate the suitability of this group of membrane probes for a particular application, we have prepared a Lipophilic Tracer Sampler Kit (L7781, DiI Derivatives for Long-Term Cellular Labeling). This kit contains 1 mg samples of nine different membrane stains, including both relatively new and well-established tracers:
- DiIC18(3) (DiI)
- DiOC18(3) (DiO)
- DiIC18(5) (DiD)
- DiIC18(7) (DiR)
- 4-Di-16-ASP (DiA)
Octadecyl rhodamine B (R18, O246) is a lipophilic cation that has been extensively used as a membrane probe (Lipid-Mixing Assays of Membrane Fusion—Note 13.1). Viral particles that have been labeled with high concentrations of R18 have fluorescence that is highly self-quenched; fusion of the particle with host cell membranes therefore results in a fusion-associated fluorescence increase.
CellMask Plasma Membrane Stains
The CellMask plasma membrane stains generate rapid and uniform cell-surface labeling without the cell type–dependent variability exhibited by lectins. CellMask Orange (C10045) and CellMask Deep Red (C10046) plasma membrane stains are optimally detected using tetramethylrhodamine (TRITC) and Cy5 dye detection configurations, respectively. They are particularly important for cell-surface demarcation in experiments using intracellular targets labeled with Green Fluorescent Protein (GFP), Alexa Fluor 488 dye, BODIPY FL dye and other green-fluorescent fluorophores. Membrane staining persists through fixation, but not permeabilization. Immunofluorescence detection of intracellular targets in cells labeled with CellMask plasma membrane stains must therefore be carried out under non-permeabilizing conditions
Fluorescent Protein–Based Plasma Membrane Markers
CellLight Plasma Membrane-CFP, CellLight Plasma Membrane--GFP and CellLight Plasma Membrane--RFP (C10606, C10607, C10608) are BacMam expression vectors encoding fusions of cyan-, green- or red-autofluorescent proteins with a plasma membrane targeting sequence consisting of the 10 N-terminal amino acids of Lck tyrosine kinase (Lck10). These fusion proteins localize to lipid rafts, providing useful alternatives to cholera toxin B conjugates (Protein Conjugates—Section 14.7) and BODIPY FL C5-ganglioside GM1 (B13950, B34401; Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3). CellLight reagents ( CellLight reagents and their targeting sequences—Table 11.1) incorporate all the customary advantages of BacMam technology, including high transduction efficiency and long-lasting, titratable expression ( BacMam Gene Delivery and Expression Technology—Note 11.1). They are provided in a ready-to-use format—simply add, incubate and image—with highly efficient expression in cell lines, primary cells, stem cells and neurons.
FM and RH Dyes
Although the lipophilic cyanine and styryl dyes described above are useful for staining cell membranes, they are relatively difficult to apply to cells and they do eventually internalize, staining all cell membranes. In contrast, FM 1-43 (T3163, T35356), FM 1-43FX (F35355), FM 4-64 (T3166, T13320), FM 4-64FX (F34653) and RH 414 (T1111) are easily applied to cells, where they bind rapidly and reversibly to the plasma membrane with strong fluorescence enhancement. These probes have large Stokes shifts and can be excited by the argon-ion laser. Because we have found that small differences in the polarity of these probes can play a large role in their rates of uptake and their retention properties, we have introduced FM 5-95 (T23360), a slightly less lipophilic analog of FM 4-64 with essentially identical spectroscopic properties.
FM 1-43 is efficiently excited with standard fluorescein optical filters but poorly excited with standard tetramethylrhodamine optical filters (). FM 1-43 has been used to outline membranes in sea urchin eggs. This styryl dye has also proven extremely valuable for identifying actively firing neurons and for investigating the mechanisms of activity-dependent vesicle cycling in widely different species (Probes for Following Receptor Binding and Phagocytosis—Section 16.1). We offer FM 1-43 in a 1 mg vial (T3163) or specially packaged in 10 vials of 100 µg each (T35356).
FM 1-43FX is an FM 1-43 analog that has been modified to contain an aliphatic amine. This modification makes the probe fixable with aldehyde-based fixatives including formaldehyde and glutaraldehyde. FM 1-43FX has been used to study synaptic vesicle cycling in cone photoreceptor terminals and to investigate the functional maturation of glutamatergic synapses. FM 1-43FX is available specially packaged in 10 vials of 100 µg each (F35355).
Membranes labeled with FM 4-64 exhibit long-wavelength red fluorescence () that can be distinguished from the green fluorescence of FM 1-43 staining () with the proper optical filter sets, thus permitting two-color observation of membrane recycling in real time. FM 4-64 selectively stains yeast vacuolar membranes and is an important tool for visualizing vacuolar organelle morphology and dynamics and for studying the endocytic pathway in yeast (Probes for Lysosomes, Peroxisomes and Yeast Vacuoles—Section 12.3). In addition, FM 4-64 staining has been used to visualize membrane migration and fusion during Bacillus subtilis sporulation—movements that can be correlated with the translocation of GFP-labeled proteins (). We offer FM 4-64 in a 1 mg vial (T3166) or specially packaged in 10 vials of 100 µg each (T13320). Additionally, we offer the fixable analog FM 4-64FX specially packaged in 10 vials of 100 µg each (F34653).
Membranes stained with RH 414 (T1111) exhibit orange fluorescence when observed through a longpass optical filter that permits passage of light beyond 510 nm. In a confocal laser-scanning microscopy study, the subcellular distribution of L-type Ca2+ channels in olfactory bulb neurons was determined using intensity ratio measurements of the green fluorescence of DM-BODIPY dihydropyridine (D7443, Probes for Ion Channels and Carriers—Section 16.3) to the red fluorescence of RH 414–stained plasma membranes (). In this method, staining of the plasma membrane by RH 414 was used to control for optical artifacts and differences in membrane surface area in the optical section. RH 414 has also been used as to follow vacuolization of the transverse tubules of frog skeletal muscle and to measure membrane potential (Probes for Membrane Potential—Chapter 22).
Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit
The Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit (I34406) provides two stains—red-fluorescent Alexa Fluor 594 wheat germ agglutinin (WGA) (excitation/emission maxima ~590/617 nm) and blue-fluorescent Hoechst 33342 dye (excitation/emission maxima when bound to DNA ~350/461 nm)—for highly selective staining of the plasma membrane and nucleus, respectively, of live, green-fluorescent protein (GFP)–transfected cells (). These dyes can be combined into one staining solution using the protocol provided, saving labeling time and wash steps while still providing optimal staining. Cell-impermeant Alexa Fluor 594 WGA binds selectively to N-acetylglucosamine and N-acetylneuraminic (sialic) acid residues. When used according to the protocol, Alexa Fluor 594 WGA provides highly selective labeling of the plasma membrane with minimal background, although labeling may not be as distinct for flat cell types when viewed using standard epifluorescence microscopy or low magnification. Alexa Fluor 594 WGA is retained after formaldehyde fixation and permeabilization with Triton X-100. This fluorescent lectin conjugate can also be used to label fixed cells; however, to avoid labeling intracellular components, formaldehyde-fixed cells should not be permeabilized before labeling. It is important to note that Alexa Fluor 594 WGA can stimulate biological activity, including clustering of glycosylated cell-surface proteins. The kit also includes Hoechst 33342 dye, a cell-permeant nucleic acid stain that is selective for DNA and is spectrally similar to DAPI. This dye does not interfere with GFP fluorescence and is retained after fixation and permeabilization. The Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit contains:
- Alexa Fluor 594 wheat germ agglutinin (WGA)
- Hoechst 33342 nucleic acid stain
- Detailed experimental protocols (Image-iT LIVE Plasma Membrane and Nuclear Labeling Kit)
Each kit provides enough staining solution for 360 assays using the protocol provided for labeling live, cultured cells that are adhering to coverslips.
Vybrant Lipid Raft Labeling Kits
The Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405) are designed to provide convenient, reliable and extremely bright fluorescent labeling of lipid rafts in live cells. Lipid rafts are detergent-insoluble, sphingolipid- and cholesterol-rich membrane microdomains that form lateral assemblies in the plasma membrane. Lipid rafts also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and receptors, which may be regulated by their selective interactions with these membrane microdomains. Recent research has demonstrated that lipid rafts play a role in a variety of cellular processes—including the compartmentalization of cell-signaling events, the regulation of apoptosis and the intracellular trafficking of certain membrane proteins and lipids —as well as in the infectious cycles of several viruses and bacterial pathogens. Examining the formation and regulation of lipid rafts is a critical step in understanding these aspects of eukaryotic cell function.
The Vybrant Lipid Raft Labeling Kits provide the key reagents for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor dyes (). Live cells are first labeled with the green-fluorescent Alexa Fluor 488, orange-fluorescent Alexa Fluor 555 or red-fluorescent Alexa Fluor 594 conjugate of cholera toxin subunit B (CT-B). This CT-B conjugate binds to the pentasaccharide chain of plasma membrane ganglioside GM1, which selectively partitions into lipid rafts. All of Molecular Probes CT-B conjugates are prepared from recombinant CT-B and are completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity. An antibody that specifically recognizes CT-B is then used to crosslink the CT-B–labeled lipid rafts into distinct patches on the plasma membrane, which are easily visualized by fluorescence microscopy. Each Vybrant Lipid Raft Labeling Kit contains sufficient reagents to label 50 live-cell samples in a 2 mL assay, including:
- Recombinant cholera toxin subunit B (CT-B) labeled with the Alexa Fluor 488 (in Kit V34403), Alexa Fluor 555 (in Kit V34404) or Alexa Fluor 594 (in Kit V34405) dye
- Anti–cholera toxin subunit B antibody (anti–CT-B)
- Concentrated phosphate-buffered saline (PBS)
- Detailed labeling protocols (Vybrant Lipid Raft Labeling Kits)
Because they are compatible with various multilabeling schemes, the Vybrant Lipid Raft Labeling Kits can also serve as important tools for identifying physiologically significant membrane proteins that associate with lipid rafts. Cells can be labeled with other live-cell probes during the lipid raft labeling protocol or immediately following the antibody crosslinking step, depending on the specific labeling requirements of the other probes. Alternatively, once the lipid rafts have been labeled and crosslinked, the cells can be fixed for long-term storage or fixed and permeabilized for subsequent labeling with antibodies or other probes that are impermeant to live cells.
Many research and biotechnological applications require detailed three- and four-dimensional visualization of embryonic cells labeled with Green Fluorescent Protein (GFP) within their native tissue environments. Fluorescent counterstains that label all the cells in a live embryo provide a histological context for the GFP-expressing cells in the specimen. The red-fluorescent CellTrace BODIPY TR methyl ester (C34556)—available separately or as part of our Image-iT LIVE Intracellular Membrane and Nuclear Labeling Kit (I34407)—is an excellent counterstain for cells and tissues that are expressing GFP. This dye readily permeates cell membranes and selectively stains mitochondria and endomembranous organelles such as endoplasmic reticulum and the Golgi apparatus, but does not appear to localize in the plasma membrane. These localization properties make the dye an ideal vital stain that can be used to reveal: (1) the location and shapes of cell nuclei, (2) the shapes of cells within embryonic tissues and (3) the boundaries of organ-forming tissues within the whole embryo. Furthermore, CellTrace BODIPY TR methyl ester staining is retained after formaldehyde fixation and permeabilization with Triton X-100, and the dye does not appear to produce any teratogenic effects on embryonic development. The emission spectra of enhanced GFP (EGFP) and CellTrace BODIPY TR methyl ester are well separated, with peaks at 508 nm and 625 nm, respectively (Figure 14.4.5), allowing simultaneous dual-channel confocal imaging without significant overspill of GFP fluorescence into the CellTrace BODIPY TR methyl ester detection channel.
The Image-iT LIVE Intracellular Membrane and Nuclear Labeling Kit provides:
- CellTrace BODIPY TR methyl ester, for staining intracellular membranes
- Hoechst 33342 dye for staining nuclei
- Detailed experimental protocols (Image-iT LIVE Intracellular Membrane and Nuclear Labeling Kit)
These two fluorescent stains were especially chosen for their compatibility with live GFP-expressing cells (), and they can be combined into one staining solution to save labeling time and wash steps while still providing optimal staining. Each kit provides enough staining solution for 250 assays using the protocol provided for labeling live, cultured cells that are adhering to coverslips.
Figure 14.4.5 Normalized absorption (—) and fluorescence emission (---) spectra of enhanced green-fluorescent protein (EGFP) and CellTrace BODIPY TR methyl ester (C34556).
The FluoroMyelin Green and FluoroMyelin Red fluorescent myelin stains (F34651, F34652) enable quick and selective labeling of myelin in brain cryosections. Visualization of myelin is useful for understanding myelin distribution, for identifying brain structures and relative location of other labels and for mapping a particular section within the brain. Traditional methods require the use of antibodies, such as anti–myelin basic protein, or chromogenic (transmitted-light) methods, such as the Loyez method or Schmued's gold chloride technique, all of which are time consuming, requiring multiple steps over one to three days. The FluoroMyelin stains, in contrast, require only a single 20-minute labeling step plus washes. A standard FITC filter set is suitable for imaging the FluoroMyelin Green stain, with little or no bleedthrough into standard DAPI, TRITC or far-red filter sets. A standard TRITC or Texas Red filter set is suitable for imaging the FluoroMyelin Red stain, with little or no bleedthrough into standard DAPI, FITC or far-red filter sets. These stains can be used in combination with antibodies and other dyes, and with standard histochemical methods for brain cryosections; they are ideal for studying demyelinating diseases such as multiple sclerosis. In addition to being used on their own, FluoroMyelin stains can be combined with other dyes in a single labeling step as in our BrainStain Imaging Kit described below, which includes NeuroTrace 530/615 red fluorescent Nissl stain and DAPI nucleic acid stain for three-color visualization.
The BrainStain Imaging Kit (B34650) enables three-color combinatorial labeling of myelin, neurons and nuclei in brain cryosections in a single 20-minute staining step plus washes (, ). This kit contains novel stains that can be used separately or together in one staining solution, replacing traditional methods that can take one to three days. Standard histochemical methods such as immunohistochemistry are compatible with these stains. Each BrainStain Kit provides:
- FluoroMyelin Green fluorescent myelin stain
- NeuroTrace 530/615 red-fluorescent Nissl stain
- DAPI nuclear counterstain
- Detailed protocols for staining mouse brain cryosections (BrainStain Imaging Kit)
|F34653||788.75||D,L||H2O, DMSO||505||47,000||725||See Notes||5, 6|
|F35355||560.09||D,L||H2O, DMSO||471||38,000||581||See Notes||5, 7|
|T3163||611.55||D,L||H2O, DMSO||471||38,000||581||see Notes||5, 7|
|T3166||607.51||D,L||H2O, DMSO||505||47,000||725||see Notes||5, 8|
|T13320||607.51||D,L||H2O, DMSO||505||47,000||725||see Notes||5, 8|
|T23360||565.43||D,L||H2O, DMSO||560||43,000||734||CHCl3||2, 6|
|T35356||611.55||D,L||H2O, DMSO||471||38,000||581||see Notes||5, 7|