Probes for the Nucleus—Section 12.5

This section describes the use of Molecular Probes nucleic acid stains for visualizing nuclei and chromosomes, as well as for analyzing chromosome banding patterns. The general chemical and spectroscopic properties of these nucleic acid stains are described in Nucleic Acid Stains—Section 8.1. The application of nucleic acid stains to the study of cell viability, cell proliferation and apoptosis is discussed in Assays for Cell Viability, Proliferation and Function—Chapter 15.

The counterstains described in this section are compatible with a wide range of cytological labeling techniques, including direct or indirect antibody-based detection methods, in situ hybridization and the detection of specific subcellular structures with fluorescent probes such as the mitochondrion-selective MitoTracker reagents (Probes for Mitochondria—Section 12.2), F-actin–selective phalloidin (Probes for Actin—Section 11.1) and autofluorescent proteins (Using Organic Fluorescent Probes in Combination with GFP—Note 12.1).

CellLight Nucleus-Targeted GFP and RFP Reagents

CellLight reagents combine the utility and selectivity of targeted fluorescent proteins with the efficiency of the BacMam gene delivery and expression technology. These reagents incorporate all the customary advantages of BacMam technology, including high efficiency transduction of mammalian cells and long-lasting, titratable expression (BacMam Gene Delivery and Expression Technology—Note 11.1). CellLight reagents 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. A complete list of CellLight reagents and their targeting sequences can be found in CellLight reagents and their targeting sequences—Table 11.1.

CellLight Nucleus-GFP (C10602), CellLight Nucleus-RFP (C10603) and CellLight Nucleus-CFP (C10616) are BacMam expression vectors encoding fusions of Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP) and Cyan Fluorescent Protein (CFP), respectively, with the SV40 nuclear localization sequence (NLS). In addition to general purpose identification and demarcation of the nucleus in live-cell imaging experiments, SV40 NLS-GFP is extensively used for analysis of nucleocytoplasmic transport and nuclear envelope integrity.

CellLight Histone 2B-GFP (C10594) and CellLight Histone 2B-RFP (C10595, Figure 12.5.1) are BacMam expression vectors encoding fusions of GFP or RFP with histone 2B. Labeling with histone 2B-GFP is a well established and minimally invasive approach for visualization of chromatin in live cells and is particularly useful for real-time imaging of mitotic cell division.

Alexa Fluor 488 Histone H1

The Alexa Fluor 488 conjugate of the lysine-rich calf thymus histone H1 (H13188) is a useful probe for nuclear protein transport assays. Nuclear-to-mitochondrial translocation of histone H1 is indicative of dsDNA strand breaks. Fluorescent histone H1 conjugates can also be used to detect membrane-surface exposure of acidic phospholipids such as phosphatidylserine.

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Cell-permeant nucleic acid stains make it possible to stain live cells or tissues that have been minimally processed. Nuclear staining can reveal the natural location of cells in tissues and can provide a means to follow nuclear changes throughout processes such as mitosis and apoptosis (Assays for Apoptosis—Section 15.5). Most of these dyes have little effect on cell function, allowing live cells to be traced as they move during development or as they infect other cells.

Cell-Permeant Blue-Fluorescent Counterstains

The membrane-permeant Hoechst 33342 dye (H1399, H3570, H21492) has been extensively used for staining the nuclei of live cells. Hoechst 33342 dye shows AT-selective staining, and Hoechst dye–stained cells and tissues show virtually no cytoplasmic staining. Hoechst 33342 is commonly used to distinguish the compact chromatin of apoptotic nuclei, in combination with BrdU labeling to identify replicating cells and to sort cells based on DNA content (Assays for Apoptosis—Section 15.5).

While not all of the blue-fluorescent cell-permeant SYTO dyes in Nucleic Acid Stains—Section 8.1 show selective nuclear staining, SYTO 40 (S11351) shows excellent staining of the nuclei in a freshwater snail embryo (). All of the blue-fluorescent SYTO dyes listed in Cell-permeant cyanine nucleic acid stains—Table 8.3 are available individually as solutions in DMSO (Nucleic Acid Stains—Section 8.1) or in a sampler kit (S11350) to facilitate finding the best counterstain for a particular cell or tissue type.

Cell-Permeant Green-Fluorescent Counterstains

Some of the green-fluorescent cell-permeant SYTO dyes (Cell-permeant cyanine nucleic acid stains—Table 8.3, Nucleic Acid Stains—Section 8.1) are excellent nuclear stains for live cells in culture () and for unfixed tissue sections. The green-fluorescent SYTO 11 dye (S7573) has shown selective nuclear staining in heart tissue, vascular endothelium and cultured myocytes and in cultured aortic vascular smooth muscle cells, showing promise for broad use in noninvasive confocal laser-scanning microscope investigations. Staining with SYTO 11 and SYTO 13 (S7575) dyes facilitated counting cells in brain slices without disrupting the three-dimensional environment. Staining with SYTO 11 dye was used to follow the movement of cells during development in whole-mount zebrafish embryos. SYTO 11 dye has also been used to identify meiotic cells in developing brain tissue.Trypanosoma cruzi stained with SYTO 11 dye can easily be detected within the cells they have infected. SYTO 13 dye was used in a double-labeling experiment with BODIPY 558/568 phalloidin (B3475, Probes for Actin—Section 11.1) to stain actin fibers, making it possible to look at nuclear changes and cytoskeletal changes concurrently in apoptotic cells.SYTO 12 dye (S7574) was used to follow chromosome movement during meiosis in live maize myocytes, and SYTO 14 dye (S7576) allowed researchers to follow RNA localization within live cells.SYTO 16 dye (S7578) served as an effective nuclear counterstain in cultured cells and has been used to stain nuclei in whole maize roots.

The green-fluorescent SYTO dyes listed in  Cell-permeant cyanine nucleic acid stains—Table 8.3 are available individually as solutions in DMSO (Nucleic Acid Stains—Section 8.1) or as components in the SYTO Green-Fluorescent Nucleic Acid Stain Sampler Kit (S7572). This SYTO Stain Sampler Kit contains 50 µL each of eight different green-fluorescent SYTO dyes to facilitate finding the best counterstain for a particular cell or tissue type.

Cell-Permeant Orange- and Red-Fluorescent Counterstains

The orange- and red-fluorescent cell-permeant SYTO nucleic acid stains listed in Cell-permeant cyanine nucleic acid stains—Table 8.3 may also prove useful as nuclear counterstains for live cells. The red-fluorescent SYTO 17 dye (S7579) was used as a nuclear counterstain for the green-fluorescent membrane stain DiOC₆(3) (D273, Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4) and with fluorescein immunostaining, as well as with the TUNEL apoptosis assay using ChromaTide fluorescein-12-dUTP (C7604, Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4) to investigate chromatin degradation and denucleation of lens tissue.Leishmania cells stained with SYTO 17 dye could later be identified in cells they had infected.SYTO 59 dye (S11341) has been used as a red-fluorescent nuclear counterstain in combination with the Green Fluorescent Protein (GFP) expressed in lymphoid cells and human embryonic kidney cells (Using Organic Fluorescent Probes in Combination with GFP—Note 12.1). SYTO 59 dye has also proven very useful in the study of Cryptosporidium oocytes because the intensity of staining appears to be related to the infectivity of the oocytes.

All of these orange- or red-fluorescent SYTO dyes are available individually as solutions in DMSO (Nucleic Acid Stains—Section 8.1) or as components in the SYTO Orange Fluorescent Stain Sampler Kit (S11360) or the SYTO Red Fluorescent Stain Sampler Kit (S11340), which contain 50 µL each of six different orange-fluorescent or seven different red-fluorescent SYTO dyes to facilitate finding the best counterstain for a particular cell or tissue type.

HCS NuclearMask and HCS CellMask Stains

In image-based high-content screening (HCS) assays, cell or object identification is the first step of automated image acquisition and analysis. For many image analysis algorithms, the cell identification process begins with the detection of fluorescently stained nuclei. Using the position of the stained nucleus as a guide, the software then extrapolates to build a mask that marks the probable position of the cytoplasmic region.

The versatile HCS NuclearMask stains, which survive standard formaldehyde-based fixation and detergent-based permeabilization methods, can be applied to live or fixed cells. For additional flexibility in multiplexing experiments, HCS NuclearMask reagents are available in three fluorescent colors (Figure 12.5.2):

• HCS NuclearMask Blue stain (excitation/emission maxima ~350/461 nm, H10325)
• HCS NuclearMask Red stain (excitation/emission maxima ~622/645 nm, H10326)
• HCS NuclearMask Deep Red stain (excitation/emission maxima ~638/686 nm, H10294)

These three HCS NuclearMask stains leave the wavelength region from 500–600 nm clear for multiplexing with green- or orange-fluorescent probes. Sufficient quantities are provided to stain ten 96-well plates using an assay volume of 100 µL per well.

In some HCS applications, cell identification based on nuclear staining alone is not adequate because the cytoplasmic region assigned by some image analysis algorithms does not accurately identify the actual cell boundaries. In addition to these HCS NuclearMask stains, we offer a series of HCS CellMask reagents that label the entire cell (i.e., cytoplasm and the nucleus) and are designed to provide an accurate backdrop against which to assess the features of interest:

• HCS CellMask Blue stain (excitation/emission maxima ~346/442 nm, H32720)
• HCS CellMask Green stain (excitation/emission maxima ~493/516 nm, H32714)
• HCS CellMask Orange stain (excitation/emission maxima ~556/572 nm, H32713)
• HCS CellMask Red stain (excitation/emission maxima ~588/612 nm, H32712)
• HCS CellMask Deep Red stain (excitation/emission maxima ~650/655 nm, H32721)

HCS CellMask stains are applied to cells immediately after fixation and permeabilization or after labeling with antibodies. Sufficient quantities are provided to stain ten 96-well plates using assay volumes of 100 µL per well.

Tracking Chromosomes through Mitosis

Many nucleic acid stains can be used to observe chromosomes caught in the act of cell division in fixed cells and tissues (, , , ). Dimeric cyanine dyes (Cell membrane-impermeant cyanine nucleic acid stains—Table 8.2, Nucleic Acid Stains—Section 8.1) have been used to observe mitotic chromosome movement in live cells. For example, YOYO-1 dye (Y3601) has been microinjected into cells in order to follow mitotic chromosomes through at least six cell cycles in fertilized sea urchin eggs (M. Terasaki and L. Jaffe, personal communication) (Figure 12.5.3).

Another useful technique for tracking chromosomes through mitosis involves metabolic incorporation of microinjected fluorescent nucleotides, including our fluorescein-12-dUTP (C7604, Labeling Oligonucleotides and Nucleic Acids—Section 8.2) by endogenous cellular enzymes into DNA. Incorporation of the fluorescent tracer does not interfere with subsequent progress through the cell cycle, and fluorescent strands of DNA can be followed as they assemble into chromosomes and segregate into daughters and granddaughters.

Blue-Fluorescent Counterstains

DAPI (D1306, D3571, D21490) is the classic nuclear and chromosome counterstain for identifying nuclei and observing chromosome-banding patterns. DAPI binds selectively to dsDNA and thus shows little to no background staining of the cytoplasm. Its relatively low-level fluorescence emission does not overwhelm signals from green- or red-fluorescent secondary antibodies or FISH probes. DAPI is semipermeant to live cells and can be used on unfixed cells or tissue sections (, ). We also offer DAPI premixed with our SlowFade, SlowFade Gold and ProLong Gold antifade reagents (S36938, S36939, P36931, P36935; Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) for simultaneous nuclear staining and antifade protection.

The Hoechst 33342 dye (H1399, H3570, H21492) has been used widely for staining the nuclei of live cells. Hoechst dyes preferentially bind to AT regions, making them quite selective (but not specific) for DNA; Hoechst dye–stained cells and tissues show virtually no cytoplasmic staining (). The Hoechst 33342 dye is commonly used in combination with labeling by 5-bromo-2'-deoxyuridine (BrdU, B23151; Labeling Oligonucleotides and Nucleic Acids—Section 8.2) to distinguish the compact chromatin of apoptotic nuclei, to identify replicating cells and to sort cells based on their DNA content (Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4, Assays for Apoptosis—Section 15.5).

The blue-fluorescent BOBO-1 nucleic acid stain (B3582) emits a brighter fluorescent signal than does DAPI. BOBO-1 has been used effectively as a counterstain for Drosophila chromosomes in combination with Cy3 dye– or fluorescein-labeled antibodies.SYTOX Blue nucleic acid stain (S11348, S34857) also emits bright blue fluorescence upon binding to nucleic acids and is a very good nuclear counterstain. Fluorescence emission of the SYTOX Blue complex with nucleic acids somewhat overlaps the emission of fluorescein, Alexa Fluor 488 and Oregon Green 488 dyes and thus we recommend SYTOX Blue dye only as a counterstain for orange- or red-fluorescent dyes.

Green-Fluorescent Counterstains

Some of our cyanine dyes ( Specialty nucleic acid reagents for molecular biology—Table 8.1, Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2, Cell-permeant cyanine nucleic acid stains—Table 8.3; Nucleic Acid Stains—Section 8.1) have been found to be useful as green-fluorescent nuclear counterstains. YO-PRO-1 dye (Y3603) and SYTOX Green stain (S7020) are excellent nuclear counterstains for cells in culture or for whole-mount tissues (, , , ) and are useful counterstains for tissue sections as well. SYTOX Green dye shows highly selective nuclear staining; YO-PRO-1 dye shows more intense staining but also weakly stains the cytoplasm and nucleolus. SYTOX Green dye has been used to follow changes in nuclear morphology in apoptotic cells () and is a component in some Molecular Probes apoptosis assay kits (Assays for Apoptosis—Section 15.5). SYTOX Green stain has been used as a specific nuclear counterstain for multicolor labeling in Drosophila imaginal disc cells. YO-PRO-1, also a component in some of Molecular Probes apoptosis assay kits (Assays for Apoptosis—Section 15.5), is selectively permeant to apoptotic cells, enabling facile identification of this cell population by flow cytometry (Figure 12.5.4). Nuclear staining by YO-PRO-1 dye has provided a method to identify individual cells within single live, perfused mesentery microvessels.

Staining with the TOTO-1 (T3600) and YOYO-1 (Y3601) nucleic acid stains enables extremely sensitive flow cytometric analysis of nuclei and isolated human chromosomes. In this study, YOYO-1 dye staining produced more than 1000 times the fluorescence signal obtained with mithramycin; furthermore, histograms of both TOTO-1 and YOYO-1 on RNase-treated nuclei provided coefficients of variation that were at least as low as those found with propidium iodide or mithramycin. These researchers also found that when nuclei were simultaneously stained with the YOYO-1 and Hoechst 33258 (H1398, H3569, H21491) dyes, the ratio of the fluorescence of these two dyes varied as a function of cell cycle. This observation suggests that our cyanine dyes might be useful for examining cell cycle–dependent changes that occur in chromatin structure. YOYO-1 dye staining also permitted the detection of discrete ribosome-containing domains within the cytoplasm of mature cell axons, which are traditionally thought to contain no transcriptional activity. In addition, YOYO-1 dye has been used as a counterstain for immunofluorescent staining of chromatin in the nuclei of developing Drosophila embryos.

Yellow-Fluorescent Counterstain

The long-wavelength tracer nuclear yellow (Hoechst S769121; , ) is often combined with the popular retrograde tracer true blue (Polar Tracers—Section 14.3) for two-color neuronal mapping. In neuronal cells, nuclear yellow primarily stains the nucleus with yellow fluorescence, whereas true blue is a UV light–excitable, divalent cationic dye that stains the cytoplasm with blue fluorescence. Both nuclear yellow and true blue are stable when subjected to immunohistochemical processing and can be used to photoconvert DAB into an insoluble, electron-dense reaction product  ( Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2).

Orange-Fluorescent Counterstains

BOBO-3 (B3586) and SYTOX Orange (S11368) cyanine dyes have fluorescence emission that is similar to that of PI, but show greater fluorescence enhancement upon binding to DNA and so should provide brighter nuclear staining. BOBO-3 dye has been used as a nuclear stain in whole-mount Xenopus laevis embryos. YOYO-3 (Y3606) and YO-PRO-3 (Y3607) dyes show strong and specific staining of the nucleus in most cultured cells.

Red-Fluorescent Counterstains

Propidium iodide (PI; P1304MP, P3566, P21493) has long been a preferred dye for red-fluorescent counterstaining of nuclei and chromosomes (). Under some fixation conditions, PI shows highly selective nuclear staining. Other preparations of cells and tissues require a simple treatment with a ribonuclease (RNase) to achieve specific nuclear staining. PI provides an excellent counterstain for cells stained with green-fluorescent probes or secondary antibodies conjugated to Alexa Fluor 488, Oregon Green, BODIPY FL or fluorescein dyes.

SYTOX Red dead cell stain (excitation/emission maxima ~640/658 nm, S34859) is a high-affinity nucleic acid stain that easily penetrates cells with compromised plasma membranes but will not cross uncompromised cell membranes. After brief incubation with SYTOX Red stain, the nucleic acids of dead cells fluoresce bright red when excited at 633 nm or 635 nm. These properties, combined with its >500-fold fluorescence enhancement upon nucleic acid binding, make the SYTOX Red stain a simple and quantitative single-step dead-cell indicator for use with red laser–equipped flow cytometers. Using 633 nm or 635 nm excitation, SYTOX Red dead cell stain is distinct from other dead cell probes like 7-AAD and PI, which are excited using 488 nm. The emission of SYTOX Red stain is limited to one channel with minimal spectral overlap, effectively freeing the channels of the 488 nm laser line.

The long-wavelength TOTO-3 (T3604) and TO-PRO-3 (T3605) dyes provide nuclear counterstains whose fluorescence is well separated from that of commonly used fluorophores, such as our popular Alexa Fluor dyes, Oregon Green, fluorescein (FITC), rhodamine (TRITC), Texas Red, coumarin (AMCA), Marina Blue and Pacific Blue dyes. Their long-wavelength spectra make these red-fluorescent nucleic acid stains particularly useful for three- or even four-color labeling using confocal laser-scanning or standard epifluorescence microscopes (, ). The absorbance peaks of the TOTO-3 and TO-PRO-3 dyes closely match the 633 nm and 635 nm laser lines of many confocal laser-scanning microscopes and the spectra match filter sets typically used for the Alexa Fluor 647 and Cy5 dyes.

Long-wavelength light–absorbing dyes have the advantage that their fluorescence is usually not obscured by the autofluorescence of tissues. For example, analysis of fluorescently stained whole-mount Xenopus laevis embryos has traditionally been difficult due to the large amount of autofluorescence from the yolk. Two reports have shown that the TO-PRO-3 dye can be used as a fluorescent nuclear stain in these embryos, allowing them to be analyzed by confocal laser-scanning microscopy. When either the 633 nm or 635 nm spectral lines of a confocal laser-scanning microscope is used with long-wavelength filter sets, the autofluorescence from the yolk is almost completely eliminated.

The TOTO-3 and TO-PRO-3 dyes were tested as counterstains for aldehyde-fixed frozen rat tissue sections. The TO-PRO-3 dye showed less cytoplasmic staining and little overlap with signals from fluorescein- or tetramethylrhodamine-labeled secondary antibodies in the same section. The TO-PRO-3 dye gives strong and selective staining of the nucleus in cultured cells. A high selectivity for nuclear staining over cytoplasmic staining made TO-PRO-3 the preferred dye for detecting amplification of the Her-2/neu gene by dual-color FISH in paraffin sections. Although its nucleic acid complex reportedly bleaches relatively rapidly, photobleaching can be slowed with antifade reagents such as are provided in our SlowFade, SlowFade Gold, ProLong and ProLong Gold antifade reagents (Fluorescence Microscopy Accessories and Reference Standards—Section 23.1). Nuclear staining by TO-PRO-3 dye has been used to study the structure of the nucleus in interphase cells  and to demonstrate segregation of chromosomes during meiosis in mouse oocytes. TO-PRO-3 dye was also used to counterstain the chromatin in nuclei of developing Drosophila embryos that were immunostained with Cy3 dye– or fluorescein-labeled antibodies. TOTO-3 dye has been used as a counterstain for TUNEL assays  and for annexin V–based apoptosis assays  (Assays for Apoptosis—Section 15.5). TOTO-3 dye has also been used in combination with Cy3 dye–labeled anti-BrdU antibody staining to show that replicons labeled with BrdU form clusters in the nucleus that are stable through several cell cycles.

SYTOX AADvanced Dead Cell Stain

Especially formulated for flow cytometry applications, SYTOX AADvanced dead cell stain (excitation/emission maxima ~546/647 nm; S10274, S10349) labels the nucleic acids of dead cells with a bright red fluorescence when excited with the 488 nm spectral line of the argon-ion laser, with minimal compensation in the green, orange and near-infrared channels. This high-affinity red-fluorescent nucleic acid stain easily penetrates cells with compromised plasma membranes, but will not cross healthy cell membranes. These properties, combined with its >500-fold fluorescence enhancement upon nucleic acid binding, make the SYTOX AADvanced dead cell stain a simple and quantitative single-step dead-cell indicator (Figure 12.5.5). Labeling of dead cells is achieved very rapidly, typically within 5 minutes. SYTOX AADvanced dead cell stain may also be useful for DNA content cell-cycle analysis with the addition of RNase A in fixed cells.

Qnuclear Deep Red Stain

Qnuclear Deep Red stain is a bright and photostable nuclear counterstain designed for use with fixed and permeabilized cells that have been labeled with Qdot nanocrystals (Qdot Nanocrystals—Section 6.6) and mounted in Qmount Qdot mounting media (Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) (Figure 12.5.6). With excitation and emission maxima of 640 and 663 nm, respectively, this counterstain can be visualized with standard fluorescence microscopy filter sets. Qnuclear Deep Red stain is compatible with Qdot 525, 565, 585, 605 and 625 nanocrystals; it can also be used with Qdot 655 nanocrystals and its conjugates, though care must be taken to use appropriate excitation wavelengths and filter sets given the fluorescence emission overlap.

SelectFX Nuclear Labeling Kit

The SelectFX Nuclear Labeling Kit (S33025) provides four spectrally distinct fluorescent dyes—blue-fluorescent DAPI, green-fluorescent SYTOX Green stain, red-fluorescent 7-aminoactinomycin D (7-AAD) and far-red–fluorescent TO-PRO-3 dye—for staining nuclei in fixed and permeabilized cells and tissues with essentially no cytoplasmic background staining. When used according to the protocol provided, the dyes in the SelectFX Nuclear Labeling Kit provide highly selective nuclear staining with little or no cytoplasmic labeling; they are ideal for use as counterstains in multicolor applications. The stained nuclei stand out in vivid contrast to other fluorescently labeled cell structures when observed by fluorescence microscopy. These dyes have excitation wavelengths that match the common laser lines for confocal microscopy and flow cytometry and can be used with standard filter sets on fluorescence microscopes and microplate readers. The staining protocol provided is compatible with a wide range of cytological labeling techniques, including direct or indirect antibody-based detection methods, mRNA in situ hybridization and staining methods that incorporate organelle- and cytoskeleton-selective fluorescent probes (including MitoTracker mitochondrion-selective probes and Alexa Fluor dye–conjugated phalloidins). The dyes can also be used to fluorescently stain cells for analysis in multicolor flow cytometry experiments. All dyes are provided as stock solutions, convenient for diluting and staining, and each dye is also available separately.

The SelectFX Nuclear Labeling Kit contains:

• DAPI, a blue-fluorescent counterstain (excitation/emission maxima ~358/451 nm)
• SYTOX Green, a green-fluorescent counterstain (excitation/emission maxima ~504/523 nm)
• 7-Aminoactinomycin D (7-AAD), a red-fluorescent counterstain (excitation/emission maxima ~546/647 nm)
• TO-PRO-3 iodide, a far-red–fluorescent counterstain (excitation/emission maxima ~642/661 nm)
• Detailed staining protocols (SelectFX Nuclear Labeling Kit)

The SelectFX Nuclear Labeling Kit contains sufficient reagents to prepare ~100 assays with each stain at 300 µL per assay.

Blue-Fluorescent Chromosome Counterstains

DAPI (D1306, D3571, D21490) is the classic blue-fluorescent nuclear and chromosome counterstain. DAPI binds selectively to dsDNA and thus shows little to no cytoplasmic background staining. DAPI's relatively low-level fluorescence emission does not overwhelm signals from green- or red-fluorescent secondary antibodies or FISH probes (Figure 12.5.7, ). We also offer DAPI premixed with our SlowFade, SlowFade Gold and ProLong Gold antifade reagents (S36938, S36939, P36931, P36935). Hoechst 33342 dye (H1399, H3570, H21492) is also commonly used for chromosome counterstaining; SYTOX Blue nucleic acid stain (S11348, S34857), which is essentially nonfluorescent except when bound to nucleic acids, may be similarly useful.

Green-Fluorescent Chromosome Counterstains

SYTOX Green (S7020) and YOYO-1 (Y3601) nucleic acid stains are useful green-fluorescent nuclear counterstains because of their bright nuclear signal and low cytoplasmic background staining. Both dyes show intense green fluorescence upon binding to nucleic acids, and a wash step is not required because the dyes are essentially nonfluorescent in aqueous medium. We have found that both SYTOX Green and YOYO-1 dyes provide simple and reliable green-fluorescent counterstains for FISH analysis, though they differ somewhat in their properties and applications. Optimal staining by the YOYO-1 dye requires RNase treatment for background reduction, whereas SYTOX Green dye staining does not. In addition, counterstaining with the SYTOX Green dye is more rapid than YOYO-1 dye counterstaining. Although the spectral properties of the two green-fluorescent dyes differ slightly, nucleic acids counterstained with either of these green-fluorescent dyes can be efficiently excited with the mercury-arc lamp or argon-ion laser and can be visualized using standard fluorescein optical filter sets.

Red-Fluorescent Chromosome Counterstains

Propidium iodide (PI; P1304MP, P3566, P21493) is the traditional red-fluorescent chromosome counterstain and can be excited with the same excitation filters used for the green-fluorescent probes. Some of our longer-wavelength cyanine dyes, including the YO-PRO-3, TO-PRO-3, YOYO-3 and TOTO-3 dyes yield red-fluorescent nuclear staining that can be excited without also exciting the fluorescence of green-fluorescent dyes. TO-PRO-3 (T3605) and TOTO-3 (T3604) dyes exhibit long–wavelength fluorescence emissions (maxima at ~660 nm) that are well separated from the emissions of other commonly used fluorophores, such as Texas Red dye, fluorescein or the Alexa Fluor dyes that absorb maximally below 600 nm (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3), making three- or even four-color labeling possible. Drosophila polytene chromosomes and nuclei of cultured mammalian cells stained with the TO-PRO-3 dye have also been detected with two-photon scanning near-field optical microscopy.

SYTOX Green Nucleic Acid Stain

Chromosomes stained with SYTOX Green dye (S7020) in combination with methyl green—a major-groove–binding dye that binds selectively to AT sequences along the chromosome—exhibit a banding pattern that indicates the location of AT-rich regions (), representing an extremely simple, rapid, fluorescence-based banding method that may prove useful for general karyotype analysis. This observation has been exploited to examine metaphase chromatin structure. The green-fluorescent SYTOX Green dye is efficiently excited by the argon-ion laser, permitting analysis of chromosome structure by confocal laser-scanning microscopy. In addition, use of SYTOX Green dye eliminates the need for RNase treatment of slides.

Acridine Homodimer

The water-soluble acridine homodimer has extremely high affinity for AT-rich regions of nucleic acids, making it particularly useful for chromosome banding. Acridine homodimer emits a blue-green fluorescence when bound to DNA, yielding fluorescence that is proportional to the fourth power of the AT base-pair content. Acridine homodimer has been recommended as an alternative to quinacrine for Q banding because of its greater brightness and higher photostability.

Other Chromosome Banding Dyes

A wide variety of fluorescent nucleic acid stains have been used for chromosome banding:

• 7-Aminoactinomycin D (7-AAD, A1310) binds selectively to GC regions of DNA, yielding a distinct banding pattern in polytene chromosomes and chromatin.
• 9-Amino-6-chloro-2-methoxyacridine (ACMA, A1324) fluoresces with greater intensity in AT-rich regions on chromosomes, yielding a staining pattern similar to the Q-banding pattern produced with quinacrine.
• DAPI () or combinations of DAPI or Hoechst 33258 (H1398, H3569, H21491) with nonfluorescent DNA-binding drugs have been used for chromosome-banding studies.
• High-resolution flow karyotyping has also been carried out with DAPI (D1306, D3571, D21490).
• Hoechst 33342 dye (H1399, H3570, H21492) has been used in chromosome sorting, multivariate analysis and karyotyping.
• Hoechst dyes have been employed in combination with chromomycin and a high-resolution, dual-laser method to sort 21 unique human chromosome types onto nitrocellulose filters, followed by hybridization to gene-specific probes.

The Nissl substance, described by Franz Nissl more than 100 years ago, is unique to neuronal cells. Composed of an extraordinary amount of rough endoplasmic reticulum, the Nissl substance reflects the unusually high protein synthesis capacity of neurons. Various fluorescent or chromophoric "Nissl stains" have been used for this counterstaining, including acridine orange, ethidium bromide,neutral red (N3246, Viability and Cytotoxicity Assay Reagents—Section 15.2), cresyl violet, methylene blue, safranin-O and toluidine blue-O. We have developed five fluorescent Nissl stains ( Fluorescence characteristics of NeuroTrace fluorescent Nissl stains—Table 14.2) that not only provide a wide spectrum of fluorescent colors for staining neurons, but also are far more sensitive than the conventional dyes:

• NeuroTrace 435/455 blue-fluorescent Nissl stain (N21479, )
• NeuroTrace 500/525 green-fluorescent Nissl stain (N21480; , , )
• NeuroTrace 515/535 yellow-fluorescent Nissl stain ()
• NeuroTrace 530/615 red-fluorescent Nissl stain (N21482; , )
• NeuroTrace 640/660 deep red–fluorescent Nissl stain (N21483)

In addition, the Nissl substance redistributes within the cell body in injured or regenerating neurons. Therefore, these Nissl stains can also act as markers for physically or chemically induced neurostructural damage. Staining by the Nissl stains is completely eliminated by pretreatment of tissue specimens with RNase; however, these dyes are not specific stains for RNA in solutions. The strong fluorescence (emission maximum ~515–520 nm) of NeuroTrace 500/525 green-fluorescent Nissl stain (N21480) makes it a good choice for use as a counterstain in combination with orange- or red-fluorescent neuroanatomical tracers such as DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4).

For a detailed explanation of column headings, see Definitions of Data Table Contents