A fast,low cost,and highly efficient fluorescent DNA labeling method using methyl green

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.     

Split-wrmScarlet and split-sfGFP: tools for faster,easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663     

灰葡萄孢菌过氧化物酶体及细胞核的荧光蛋白标记

【目的】对灰葡萄孢菌(Botrytis cinerea)的细胞核和过氧化物酶体进行荧光蛋白标记,为研究其生长发育和侵染过程中细胞结构和细胞器动态提供基础。【方法】以绿色荧光蛋白(GFP)和红色荧光蛋白(DsRED、mCherry)为报告基因,利用根癌农杆菌介导转化(Agrobacterium tumefaciens mediated transformation,AtMT)将3种荧光蛋白标记载体分别导入灰葡萄孢菌标准菌株B05.10;通过PCR检测及荧光观察筛选和验证转化子,并进行单孢纯化;利用共聚焦显微镜记录细胞器荧光定位情况。【结果】获得了过氧化物酶体或细胞核稳定表达红、绿色荧光的重组单孢菌株,PCR验证表明标记基因成功整合入转化子基因组。在标记细胞核的菌株中,菌丝和孢子中可见多个明亮、圆形的荧光点,与DAPI染色共定位。标记过氧化物酶体的菌株中,菌丝和孢子中可见小点状绿色或红色荧光,在脂类物质诱导下荧光点数量明显增加,符合过氧化物酶体分布及动态特征。细胞壁染色结果显示,细胞壁染色产生的蓝色荧光与红、绿荧光蛋白的荧光互不干扰,标记效果良好。【结论】获得了理想的过氧化物酶体或细胞核荧...     

Visualization of viral DNA assembly using nonfluorescent staining

ABSTRACT

We present an easy test for rapid visualization of viral DNA assemblies in infected cell cytoplasm. We selected the best stains for nuclear staining: Nile blue A, Bismarck brown, gallocyanin chrome alum, methyl green pyronin and azure II. None of the staining techniques is fluorescent, which facilitates their use in everyday experiments. Methyl green is most promising for routine detection of viral DNA assemblies in the cytoplasm; the procedure enables ready detection of viral DNA accumulation in the cytoplasm.     

An improved formulation of SYPRO Ruby protein gel stain: comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix-assisted laser desorption/ionisation-time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post-staining gels subjected to labeling with other fluorophores such as Schiff's base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post-staining gels after detection of glycoproteins using the green fluorescent Pro-Q Emerald 300 glycoprotein stain or detection of beta-glucuronidase using the green fluorescent ELF 97 beta-D-glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.     

Chemical methods of DNA and RNA fluorescent labeling.

Several procedures have been described for fluorescent labeling of DNA and RNA. They are based on the introduction of aldehyde groups by partial depurination of DNA or oxidation of the 3'-terminal ribonucleoside in RNA by sodium periodate. Fluorescent labels with an attached hydrazine group are efficiently coupled with the aldehyde groups and the hydrazone bonds are stabilized by reduction with sodium cyanoborohydride. Alternatively, DNA can be quantitatively split at the depurinated sites with ethylenediamine. The aldimine bond between the aldehyde group in depurinated DNA or oxidized RNA and ethylenediamine is stabilized by reduction with sodium cyanoborohydride and the primary amine group introduced at these sites is used for attachment of isothiocyanate or succinimide derivatives of fluorescent dyes. The fluorescent DNA labeling can be carried out either in solution or on a reverse phase column. These procedures provide simple, inexpensive methods of multiple DNA labeling and of introducing one fluorescent dye molecule per RNA, as well as quantitative DNA fragmentation and incorporation of one label per fragment. These methods of fluorophore attachment were shown to be efficient for use in the hybridization of labeled RNA, DNA and DNA fragments with oligonucleotide microchips.     

Methyl green staining and highly repetitive DNA in polytene chromosomes

Fixed polytene chromosomes of Rhynchosciara angelae were stained with methyl green-pyronin after different pretreatments. Methyl green affinity for chromatin in the polytene chromosomes is lost after acid, alkali and heatformaldehyde treatments as a consequence of DNA denaturation, but reappears if conditions for DNA strand reassociation are provided. When such conditions are absent, only some chromosomal regions recover their affinity for methyl green. The resulting differential staining pattern is described and its possible relation to highly repetitive DNA sequences is discussed.     

Changes in retention behavior of fluorescently labeled proteins during ion-exchange chromatography caused by different protein surface labeling positions

Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.     

Monitoring lysosomal fusion in electrofused hybridoma cells

Dendritic and tumor cells are fused to produce hybridoma cells, which are considered to be used as cellular vaccines to treat cancer. Previous strategies for hybridoma cell production were based on the quantification of the electrofusion yield by labeling the cytoplasm of both parental cell types. However, a better physiological strategy would be to label subcellular structures related directly to the antigen presentation process. Therefore, we here electrofused the same amount of CHO cells stained with red and green fluorescent dextrans and have monitored the yield of hybridoma cell formation by measuring the fusion of red and green late endocytic organelles that are involved in antigen presentation. By using confocal microscopy, the level of fused, fluorescently labelled late endocytic compartments in a single hybridoma cell was determined. The results demonstrate that organellar fusion occurs in hybridomas, which is time- and temperature-dependent. This approach therefore provides a new method for the hybridoma cell vaccine evaluation, which is based on the intracellular physiological mechanism of antigen presentation.     

Methyl green-pyronin; stoichiometry of reaction with nucleic acids

Study of the stoichiometry of the DNA-methyl green reaction by dialysis, precipitation of stain-nucleic acid mixtures, and the staining of nuclei of known DNA content, indicate that the compound consists of one dye molecule per 10 P. The significance of this result was discussed in the preceding paper (1). Histone and lanthanum (and probably other multivalent cations (3)) compete with the dye for the nucleic acid molecule, indicating a common site of attachment, presumably the phosphoric acid groups. With care in the avoidance of procedures which might depolymerize DNA, and the use of a buffer at about pH 4.1, a quantitative histochemical method for DNA by the use of methyl green is possible. Pyronin staining appears to be of qualitative significance only. Slight differences in degree of polymerization, as between the shad and mammalian DNA appear to have no effect on methyl green staining. It may be that a critical level of polymerization for DNA staining exists. This level must exceed 20 nucleotides to account for the 10 P to 1 dye molecule and the effect on the methyl green absorption spectrum; but it may be considerably greater. Beyond this critical level, whatever it may be, further polymerization probably has no influence on staining.     

MRI Detection of Nonproliferative Tumor Cells in Lymph Node Metastases Using Iron Oxide Particles in a Mouse Model of Breast Cancer

Cell tracking with magnetic resonance imaging (MRI) and iron nanoparticles is commonly used to monitor the fate of implanted cells in preclinical disease models. Few studies have employed these methods to study cancer cells because proliferative iron-labeled cancer cells will lose the label as they divide. In this study, we evaluate the potential for retention of the iron nanoparticle label, and resulting MRI signal, to serve as a marker for slowly dividing cancer cells. Green fluorescent protein-transfected MDA-MB-231 breast cancer cells were labeled with red fluorescent micron-sized superparamagnetic iron oxide (MPIO) nanoparticles. Cells were examined in vitro at multiple time points after labeling by staining for iron-labeled cells and by flow cytometric detection of the fluorescent MPIO. Severe combined immune deficiency (SCID) mice were implanted with 5 x 10⁵ MPIO-labeled or unlabeled cells in the mammary fat pad and MRI was performed weekly until 28 days after injection. Microscopy was performed to validate MRI. In vitro assays revealed a very small percentage of cells that retained MPIO at 14 days after labeling. Regions of signal loss were observed in MRI of primary tumors that developed from iron-labeled cancer cells. Small focal regions of signal loss were detected in images of the axillary and brachial nodes in six of eight mice, at day 14 or later, with microscopy confirming the presence of iron-labeled cancer cells. Our data suggest an interesting role for cell tracking with iron particles since label retention leads to persistent signal void, allowing proliferative status to be determined.     

A new rapid method for selective extraction of RNA from fixed mammalian tissues.

Treatment of formalin-fixed mammalian tissues with concentrated or 50% phosphoric acid at 5 degrees C for 20 and 50 min. respectively reveals complete extraction of RNA as judged by methyl green followed by staining with pyronin. This procedure also causes depolymerisation of DNA as indicated by the red staining of the nuclei. Sections treated with concentrated phosphoric acid at 5 degrees C for 30 min. causes disruption of the double helical structure of DNA what results in the depression of the pyronin staining. Similarly treated sections show Feulgen positive nuclei. Treatment of sections in 25 % phosphoric acid at 60 degrees C for 15 min. followed by staining with methyl green and pyronin show red nuclei, nucleoli and the cytoplasm. This indicates that extraction of RNA is only possible in cold and not at elevated temperature.     

A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling

We have cloned a gene which encodes a fluorescent protein from the stony coral, Galaxeidae. This protein absorbs light maximally at 492 nm and emits green light at 505 nm, and as a result, we have designated it "Azami-Green (AG)." Despite sharing a similar spectral profile with enhanced green fluorescent protein (EGFP) (Clontech), the most popular variant of the Aequorea victoria green fluorescent protein, the identity between these two proteins at the amino acid level is only 5.7%. However, since AG has a high extinction coefficient, fluorescence quantum yield, and acid stability, it produces brighter green fluorescence in cultured cells than EGFP. Similar to other fluorescent proteins isolated from coral animals, AG forms a tight tetrameric complex, resulting in poor labeling of subcellular structures such as the plasma membrane and mitochondria. We have converted tetrameric AG into a monomeric form by the introduction of three amino acid substitutions, which were recently reported to be effective for monomerizing the red fluorescent protein from Discosoma coral (DsRed, Clontech). The resultant monomeric AG allowed for efficient fluorescent labeling of all of the subcellular structures and proteins tested while retaining nearly all of the brightness of the original tetrameric form. Thus, monomeric AG is a useful monomeric green-emitting fluorescent protein comparable to EGFP.     

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.     

Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines.     

Improved staining method for the simultaneous flow cytofluorometric analysis of DNA content, S-phase fraction, and surface phenotype using single laser instrumentation.

We have developed an improved technique for triple staining that permits the simultaneous flow cytofluorometric analysis of cell surface antigens, bromodeoxyuridine incorporation into DNA, and DNA quantification using 7-amino-actinomycin D. PHA-activated human peripheral blood lymphocytes were incubated with bromodeoxyuridine and stained for cell surface phenotype with phycoerythrin-labeled monoclonal antibodies. Stained cells were fixed serially with 1% paraformaldehyde and 45% ethanol. Fixed cells were sequentially stained with an anti-BrdUrd monoclonal antibody followed by a FITC-conjugated goat anti-mouse antibody and incubated with 7-amino-actinomycin D. Hypotonic buffer was employed for all procedures after fixation. Stained-fixed cells were analyzed by flow cytofluorometry for simultaneous green (525 nm), orange (570 nm), and red (greater than 650 nm) fluorescence. Utilizing this staining technique, we were able to analyze simultaneously cell phenotype, DNA synthesis, and total cellular DNA content with single laser excitation.     

Basic Two-Dye Stains for Epoxy-Embedded 0.3-1 μ Sections

Dyes used in the 3 methods recommended are: I, thionin and acridine orange (T-AO); II, Janus green and Darrow red (JG-DR); III, methyl green and methyl violet (MG-MV). The first 2 methods were two-solution stains, applied in sequence; the third, required only one solution since methyl violet is present in commercial methyl green. Staining solution and timing was as follows: Method I. 0.1% thionin in a 45% ethanolic solution of 0.01 N NaOH, 5 min at 70 C; rinsing in water and followed by 1 min in a 1% aqueous solution of acridine orange made up in 0.02 N NaOH, also at 70 C, then washed, and dried on slides. Method II. 0.5% Janus green in aqueous 0.05 N NaOH, 5 min at 70 C; rinsing in water then into 0.5% Darrow red in 0.05 N NaOH (aq.), 2 min at 70 C., washing, and drying on slides. Method III. 1% methyl green (commercial, unpurified) in 1% aqueous borax for 15-20 min at 20-25 C, washing and attaching to slides. All staining was performed by floating the sections on the staining solutions, all drying at 70 C, and mounting in a resinous medium. T-AO gave blue to violet cytoplasmic structures, darker nuclei which contrasted strongly with yellow connective tissue and the secretion of goblet cells. JG-DR resembled a hematoxylineosin stain, but by shortening the staining time in DR to 0.5-1 min, collagenous and elastic tissue retained more of the green dye. MG-MV gave dark green nuclei in light green cytoplasm, with collagenous and elastic tissues in blue to violet. As with most methods for staining ultrathin sections, thicknesses of less than 1 μ required longer staining times.     

A single laser method for subtraction of cell autofluorescence in flow cytometry

In flow cytometry cell autofluorescence often interferes with efforts to measure low levels of bound fluorescent antibody. We have developed a way to correct for autofluorescence on a cell-by-cell basis. This results in improved estimates of real staining and better separation of the fluorescence histograms of stained and non-stained cells. Using a single laser, two-color fluorescence measurement system and two-color compensation electronics, autofluorescence and one fluorescent reagent are measured (rather than two fluorescent reagents). With fluorescein-conjugated antibodies the signal in the 515 to 555 nm range (green fluorescence) includes both fluorescein emission and part of the cellular autofluorescence. In the cases we have investigated, autofluorescence collected at wavelengths above 580 nm ("red") is well correlated with the green autofluorescence of the cells. A fraction of this red fluorescence is subtracted from the green fluorescence to produce an adjusted fluorescein output on which unstained cells have zero average signal. Use of this method facilitates the selection of rare cells transfected with surface antigen genes. Culture conditions affect the level of autofluorescence and the balance between red and green autofluorescence. When applied with fluorescein-conjugated reagents, the technique is compatible with the use of propidium iodide for live/dead cell discrimination.     

A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction.

We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.