Nuclear and Chromosome Counterstaining and Nissl Stains - Section 8.6

Blue-Fluorescent Counterstains

DAPI (D1306, D3571, D21490) is the classic nuclear and chromosome counterstain, used for years to identify nuclei and show 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 Light and ProLong Gold antifade reagents (S24635, S24636, P36931, P36935) for simultaneous nuclear staining and antifade protection.

The Hoechst 33342 dye (H1399, H3570, H21492) has been used widely for staining the nuclei of living 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) 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).

Our Patented 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. The SYTOX Blue nucleic acid stain (5 mM solution in dimethylsulfoxide (DMSO), S11348; 1 mM solution in DMSO, 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 () and our 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 proprietary cyanine dyes (Nucleic Acid Stains—Section 8.1; 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) provide the only green-fluorescent nuclear counterstains available. Our YO-PRO-1 dye (Y3603) and SYTOX Green stain (S7020) are excellent nuclear counterstains for cells in culture or for whole-mount tissues (, , , ) and will most likely be useful counterstains for tissue sections as well. The SYTOX Green dye shows highly selective nuclear staining; the YO-PRO-1 dye shows more intense staining but also weakly stains the cytoplasm and nucleolus. The SYTOX Green dye has been used to follow changes in nuclear morphology in apoptotic cells () and is a component in our Vybrant Apoptosis Assay Kit #1, #8, #9 and #10 (V13240, V35112, V35113, V35114; Assays for Apoptosis—Section 15.5). The SYTOX Green stain has been used as a specific nuclear counterstain for multicolor labeling in Drosophila imaginal disc cells. Nuclear staining by the YO-PRO-1 dye has provided a method to identify individual cells within single living, perfused mesentery microvessels. YO-PRO-1, which is a component of our Vybrant Apoptosis Assay Kit #4 and #7 (V13243, V23201; Assays for Apoptosis—Section 15.5), and several of our other "cyanine monomer" series of nucleic acid stains (Nucleic Acid Stains—Section 8.1, Cell membrane-impermeant cyanine nucleic acid stains—Table 8.2) is selectively permeant to apoptotic cells, enabling facile identification of this cell population by flow cytometry (Assays for Apoptosis—Section 15.5, Figure 15.85). Our YOYO-1 dye (Y3601) has been used as a counterstain for immunofluorescent staining of chromatin in the nuclei of developing Drosophila embryos. The high sensitivity of YOYO-1 dye staining permitted the detection of discrete ribosome-containing domains within the cytoplasm of mature cell axons, which are traditionally thought to contain no transcriptional activity.

Figure 15.85 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #4 (V13243). Jurkat human T-cell leukemia cells were first exposed to10 µM camptothecin for four hours (top panel) or left untreated (as control, bottom panel). Cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #4 and analyzed by flow cytometry. Note that the camptothecin-treated cells (top panel) have a significantly higher percentage of apoptotic cells (indicated by an "A") than the basal level of apoptosis seen in the control cells (bottom panel). V = viable cells, D = dead cells.

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.

Yellow-Fluorescent Counterstain

The long-wavelength tracer nuclear yellow (Hoechst S769121, N21485; , ) is often combined with the popular retrograde tracer true blue (T1323, 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- and Red-Fluorescent Counterstains

Propidium iodide (PI; P1304MP, P3566, P21493) has long been the 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.

The 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. The BOBO-3 dye has been used as a nuclear stain in whole-mount Xenopus laevis embryos. Our YOYO-3 (Y3606) and YO-PRO-3 (Y3607) dyes show strong and specific staining of the nucleus in most cultured cells.

Long-Wavelength Nuclear Counterstains

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 647 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. The high absorptivity of TO-PRO-3 is sufficient to make it a good chromosome stain, even when excited at 488 nm.

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 647 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 Light, ProLong and ProLong Gold antifade reagents (S2828, S7461, P7481, P36930, P36934; Fluorescence Microscopy Accessories and Reference Standards—Section 23.1). Nuclear staining by the 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. The 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. Our 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). The 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. Molecular Probes provides monoclonal anti-BrdU antibody labeled with the spectrally similar Alexa Fluor 546 dye (A21308, Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4) and with several other Alexa Fluor dyes for cell proliferation and apoptosis studies (Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4).

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.

Cytological Nuclear Counterstain Kit

For RNA in situ hybridization with tissue culture cells, we recommend our Cytological Nuclear Counterstain Kit (C7590), which contains three spectrally distinct fluorescent dyes for staining nuclei in fixed-cell preparations:

• Concentrated SYTOX Green nucleic acid stain in DMSO
• Concentrated propidium iodide in water
• Concentrated DAPI in water
• Detailed protocols for fixation and staining (Cytological Nuclear Counterstain Kit)

Each of the nucleic acid stains is provided as a convenient 300X solution, ready for dilution and staining. This kit provides sufficient reagents for staining 300 slides with each counterstain.

The reagents in the Cytological Nuclear Counterstain Kit are especially valuable for multicolor applications, in which an appropriate counterstain can be selected to contrast spectrally with other fluorescent probes in the sample. When used according to our protocols, the counterstains in this kit selectively stain nuclei, with little or no cytoplasmic labeling. Observed by fluorescence microscopy, the nuclei stand out in vivid contrast to fluorescent probes of other cell structures ().

Cell-permeant nuclear 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 meiosis 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. In particular, some of our SYTO dyes have greatly facilitated the studies of live cells.

Cell-Permeant Blue-Fluorescent Counterstains

The membrane-permeant Hoechst 33342 dye (H1399, H3570, H21492) has been extensively used for staining the nuclei of living cells. The Hoechst 33342 dye shows AT-selective staining — 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 SYTO dyes in Nucleic Acid Stains - Section 8.1 show specific 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 or in a sampler kit (S11350, SYTO(R) Blue Fluorescent Nucleic Acid Stains) to facilitate finding the best counterstain for a particular cell or tissue type.

Cell-Permeant Green-Fluorescent Counterstains

Some of our predominantly green-fluorescent cell-permeant SYTO dyes (Nucleic Acid Stains - Section 8.1, Cell-permeant cyanine nucleic acid stains - Table 8.3) 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. For instance, use of the SYTO 11 and SYTO 13 (S7573, S7575; Nucleic Acid Stains - Section 8.1) dyes facilitated counting cells in brain slices without disrupting the three-dimensional environment. Staining with the SYTO 11 dye was used to follow the movement of cells during development in whole-mount zebrafish embryos. The SYTO 11 dye has also been used to identify meiotic cells in developing brain tissue. Trypanosoma cruzi whose nuclei have been stained with the SYTO 11 dye can easily be detected in cells they have infected. Our SYTO 16 dye (S7578) is also an effective nuclear counterstain in cultured cells and has been used to stain nuclei in whole maize roots.

The green-fluorescent SYTO dyes (Product Information Sheet) have also been used to investigate changes in the nucleus in live cells. For example, the 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. The SYTO 12 dye (S7574) was used to follow chromosome movement during meiosis in live maize myocytes, and the SYTO 14 dye (S7576) allowed researchers to follow RNA localization within living cells.

The green-fluorescent SYTO dyes listed in Cell-permeant cyanine nucleic acid stains - Table 8.3 are available individually dissolved in either 100 µL or 250 µL of DMSO, as components in one of two SYTO Green-Fluorescent Nucleic Acid Stain Sampler Kits (S7554, S7572; SYTO(R) Green-Fluorescent Nucleic Acid Stains) or in the RediPlate 96 nucleic acid stain sampler microplate (R32715, Nucleic Acid Stains - Section 8.1). These SYTO Stain Sampler Kits contain 50 µL each of six different green-fluorescent SYTO dyes to facilitate finding the best counterstain for a particular cell or tissue type. The RediPlate 96 nucleic acid stain sampler microplate facilitates the screening of a wide array of nucleic acid–binding dyes — including 36 different SYTO dyes, SYBR Green I, SYBR Green II and PicoGreen dyes, amine-reactive SYBR 101 and SYBR 103 dyes, SYTOX Green, SYTOX Orange and SYTOX Blue dyes, Hoechst 33342, propidium iodide and hexidium iodide — that have been predispensed into a 96-well microplate.

Cell-Permeant Orange- and Red-Fluorescent Counterstains

The red-fluorescent SYTO 17 dye (S7579) was used as a nuclear counterstain for the green-fluorescent membrane stain DiOC₆(3) (D273, Probes for Mitochondria - Section 12.2), with fluorescein immunostaining, and with the TUNEL apoptosis assay using ChromaTide fluorescein-12-dUTP (C7604, Labeling Oligonucleotides and Nucleic Acids - Section 8.2) to investigate chromatin degradation and denucleation of lens tissue. Leishmania cells stained with the SYTO 17 dye could later be identified in cells they had infected. Our 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 (Fluorescent Probes for Use with GFP - Note 12.1). SYTO 59 dye has also proven very useful in the study of Cryptosporidium oocytes; the intensity of staining appears to be related to the infectivity of the oocytes, providing a valuable research tool.

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

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, Dimeric Cyanine Nucleic Acid Stains) have also 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 (Figure 8.138). Another useful technique for tracking chromosomes through mitosis involves metabolic incorporation of microinjected fluorescent nucleotides, including our fluorescein-12-dUTP, Oregon Green 488-5-dUTP and BODIPY TR-14-dUTP (C7604, C7630, C7618; 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. Presumably, injection of 5'-bromo-2'-deoxyuridine triphosphate (BrdUTP, B21550; Labeling Oligonucleotides and Nucleic Acids - Section 8.2), followed by detection of the incorporated BrdU with one of our Alexa Fluor conjugates of the anti-BrdU antibody (Assays for Cell Enumeration, Cell Proliferation and Cell Cycle - Section 15.4), would also be suitable for studying mitosis.

Figure 8.138 Using the YOYO-1 dye to follow cell division in a sea urchin egg. The YOYO-1 dye (Y3601) was injected into an unfertilized sea urchin egg. The egg was fertilized and then observed by confocal laser-scanning microscopy. Images were obtained every 15 sec in this sequence. Every fourth image is shown in the first part, then every image is shown during chromosome separation. The image was contributed by Mark Terasaki, University of Connecticut Health Center.

Molecular Probes provides a spectrum of dyes for counterstaining hybridized metaphase or interphase chromosomes in fluorescence in situ hybridization (FISH) assays (Detecting Nucleic Acid Hybridization—Section 8.5). Our extensive selection of counterstains in colors spanning the spectrum makes it easy to find the ideal counterstain.

Red-Fluorescent Counterstaining for Green-Fluorescent Probes

The best contrast provided for one-color staining with green-fluorescent antibodies or probes is a red-fluorescent counterstain. Green-fluorescent signals appear yellow when overlapping the red counterstaining (). Propidium iodide (PI; P1304MP, P3566, P21493) is a preferred red-fluorescent nuclear counterstain that can be excited using excitation filters appropriate for green-fluorescent dyes. Staining by PI also yields relatively low background fluorescence from RNA staining. Some of our longer-wavelength cyanine dyes, including the YO-PRO-3, TO-PRO-3, YOYO-3 and TOTO-3 dyes (see below) yield red-fluorescent nuclear staining that can be excited without exciting the fluorescence of green-fluorescent dyes.

The Best Green-Fluorescent Counterstains

The SYTOX Green (S7020) and YOYO-1 (Y3601) nucleic acid stains are the premier dyes for green-fluorescent nuclear counterstains because of their bright nuclear signal and low cytoplasmic background staining. Both dyes show bright green fluorescence upon binding to nucleic acids and a wash step is not required because the dyes are essentially nonfluorescent in aqueous medium. Researchers at Molecular Probes have found that both the YOYO-1 and SYTOX Green dyes provide simple and reliable green-fluorescent counterstains for FISH analysis (Detecting Nucleic Acid Hybridization—Section 8.5), 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. Dual-wavelength excitation of either TOTO-1 dye– or YOYO-1 dye–stained chromosomes reportedly permits specific chromosomes to be identified and sorted.

Blue-Fluorescent Counterstaining for Multicolor Labeling

DAPI (D1306, D3571, D21490) is the classic nuclear and chromosome counterstain, used for years to identify nuclei and show chromosome-banding patterns (). 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 8.86, ). We also offer DAPI premixed with our SlowFade, SlowFade Light and ProLong Gold antifade reagents (S24635, S24636, P36931, P36935). The Hoechst 33342 dye (H1399, H3570, H21492) is also commonly used for chromosome counterstaining. Our SYTOX Blue nucleic acid stain (S11348, S34857), which is essentially nonfluorescent except when bound to nucleic acids, may be similarly useful.

Figure 8.86 Fluorescence in situ hybridization (FISH) mapping of a BAC clone on human metaphase chromosomes. FISH was performed using a BAC clone labeled using the ARES Alexa Fluor 488 DNA Labeling Kit (A21665). The chromosomes were counterstained with DAPI (D1306, D3571, D21490). Image contributed by Nallasivam Palanisamy, Cancer Genetics Inc.

Very Long-Wavelength Counterstains for Multicolor Labeling

Our Patented TO-PRO-3 (T3605) and TOTO-3 (T3604) dyes are excellent chromosomal counterstains that are particularly useful for multicolor labeling. These dyes have very 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 family of 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

When chromosomes are stained with the SYTOX Green dye in combination with methyl green — a major-groove–binding dye that binds selectively to AT sequences along the chromosome — a banding pattern arises that indicates the location of AT-rich regions (). This phenomenon has been exploited to examine metaphase chromatin structure and represents an extremely simple, rapid, fluorescence-based banding method that may prove useful for general karyotype analysis. 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 the SYTOX Green dye eliminates the need for RNase treatment of slides.

Acridine Homodimer

The water-soluble acridine homodimer (A666) 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 Dyes and Chromosome Banding Reagents

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

• Hoechst 33342 dye (H1399, H3570, H21492) has been used in chromosome sorting, multivariate analysis and karyotyping.
• High-resolution flow karyotyping has also been carried out with DAPI (D1306, D3571, D21490).
• DAPI () or combinations of DAPI or Hoechst 33258 (H1398, H3569, H21491) with nonfluorescent DNA-binding drugs have been used for chromosome-banding studies.
• The 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.
• 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.

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 (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 (N21481, )
• 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 the physiological state of the neuron. 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 the preferred dye 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).