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Histochemistry and Cell Biology

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

Journal:For Histochemistry Peer and CellReview Biology Manuscript ID: HCB-2855-13-Drenckhahn.R1

Manuscript Type: Original manuscripts

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Complete List of Authors: Prieto, Daniel; Institut Pasteur de Montevideo, ; Facultad de Ciencias, Universidad de la República, Biología Celular Aparicio, Gonzalo; Facultad de Ciencias, Universidad de la República, Biología Celular Morande, Pablo; Institut Pasteur de Montevideo, Zolessi, Flavio; Facultad de Ciencias, Universidad de la República, Biología Celular; Institut Pasteur de Montevideo,

methyl green, DNA, flow cytometry, confocal microscopy, electrophoresis, Keywords: fluorescence

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Page 1 of 65 Histochemistry and Cell Biology

1 2 3 A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl 4 5 green. 6 7 8 Daniel Prieto 1,2 , Gonzalo Aparicio 1, Pablo E. Morande 2 and Flavio R. Zolessi 1,2 9 10 11 12 13 14 1. Sección Biología Celular, Facultad de Ciencias, Universidad de la República, Uruguay 15 16 17 Address: Iguá 4225, Montevideo, 11400 Uruguay 18 For Peer Review 19 20 2. Institut Pasteur de Montevideo, 11400 Uruguay 21 22 23 Address: Mataojo 2020, Montevideo, Uruguay 24 25 26 27 28 29 Corresponding Author: Dr. Flavio R. Zolessi 30 31 32 Email: [email protected] 33 34 35 Phone/Fax: (+598) 2522 0910 36 37 38 39 40 41 Keywords: methyl green, DNA, flow cytometry, confocal microscopy, electrophoresis, 42 43 fluorescence 44 45 46 Abstract word count: 225 47 48 49 Full-text word count: 4990 50 51 52 53 54 55 56 57 58 59 60 1

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1 2 3 ABSTRACT 4 5 6 The increasing need for multiple-labeling of cells and whole organisms for fluorescence 7 8 microscopy has led to the development of hundreds of fluorophores that either directly 9 10 recognize target molecules or organelles, or are attached to antibodies or other 11 12 13 molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as 14 15 well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH 16 17 or cytometry. However, there are currently few reliable red to far-red emitting DNA 18 For Peer Review 19 stains that can be used. We describe herein an extremely simple, inexpensive and robust 20 21 22 method for DNA labeling of cells and electrophoretic gels using the very well-known 23 24 histological stain methyl green (MG). MG used in very low concentrations at 25 26 physiological pH proved to have relatively narrow excitation and emission spectra, with 27 28 peaks at 633 and 677 nm respectively, and a very high resistance to photobleaching. It 29 30 can be used in combination with other common DNA stains or antibodies without any 31 32 33 visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a 34 35 similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we 36 37 show here that MG fluorescence can be used as a stain for direct measuring of viability 38 39 by both microscopy and flow cytometry, with full correlation to ethidium bromide 40 41 42 staining. MG is thus a very convenient alternative to currently used red-emitting DNA 43 44 stains. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2

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1 2 3 INTRODUCTION 4 5 6 Microscopy imaging has been a pillar of biological research since the invention of the 7 8 first microscopes, and in the post-genomic era it has clearly become an essential tool for 9 10 the further advancement in the full molecular characterization of cells and organisms. 11 12 13 Fluorescence microscopy, and in particular laser scanning confocal microscopy (LSCM), 14 15 is routinely used for most molecular studies on biological processes. This has been 16 17 aided by the relatively recent development of multiple fluorophores specially designed 18 For Peer Review 19 for laser excitation and long-lasting fluorescence emission (Panchuk-Voloshina et al. 20 21 22 1999; Terai and Nagano 2013). Many of these compounds are, however, relatively 23 24 expensive. 25 26 27 DNA fluorescent staining is widely used for routine counterstaining of tissues and cells, 28 29 as well as for the detailed study of nuclear and chromosomal structure for research and 30 31 clinical diagnosis (Klonisch et al. 2010). Although several DNA stains are currently 32 33 available, traditionally the most widely used have been those excited by UV light and 34 35 36 emitting in blue, like DAPI or Hoechst minor groove-binding agents (Kapuscinski 1995; 37 38 Latt and Stetten 1976). The reason for this is that these fluorophores fit in the usual 39 40 three-filter systems used in most conventional epifluorescence microscopes, being 41 42 typically combined with green and orange-red emitting fluorophores that can be easily 43 44 45 attached to antibodies or other molecules (like FITC and TRITC, respectively). The 46 47 progressive incorporation of spectral detection in LSCMs, combined with a wide variety 48 49 of laser emission lines, has allowed researchers to choose for the use of far-red-emitting 50 51 nuclear stains such as propidium iodide (PI) or TO-PRO 3 (Van Hooijdonk et al. 1994). 52 53 54 In addition, UV lasers are relatively expensive, and it is rather common for institutions 55 56 to have LSCMs not equipped for blue fluorophore detection. Another problem faced by 57 58 microscopists is that many cells and tissues, particularly in embryos, contain 59 60 3

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1 2 3 autofluorescence emitting in a wide range of wavelengths, from blue to orange. Far red 4 5 fluorophores have helped to overcome this problem (Beumer et al. 1995). 6 7 8 Methyl green (MG) is a triphenylmethane dye which has been widely used as a nuclear 9 10 colored stain for histological sections since the late 19th century (Høyer et al. 1986) and 11 12 13 for biochemical analysis of DNA degradation (De Petrocellis and Parisi 1973; Kurnick 14 15 and Sandeen 1960). It binds to DNA, but not RNA, in a non-intercalating manner, 16 17 interacting with the major groove and showing preferential binding for AT-rich regions 18 For Peer Review 19 (Kim and Nordén 1993; Krey and Hahn 1975; Kurnick 1952; Kurnick and Foster 1950). 20 21 22 Although its field of application has been mostly restricted to brightfield microscopy, 23 24 some reports suggest its use in fluorescent staining protocols for microscopy as a 25 26 quencher of DNA fluorescence (Li et al. 2003). As MG was shown not to bind RNA, it 27 28 has also been used as a complement for RNA determination by Pironin Y in flow 29 30 cytometry, because it blocks the non-specific binding of this dye to DNA (Pollack et al. 31 32 33 1982). 34 35 36 In a search for a rapid and stable nuclear staining for confocal microscopy in the red/far 37 38 red region of the visible spectrum, we found that an extremely diluted aqueous MG 39 40 solution at physiological pH provided a suitable and extremely convenient alternative to 41 42 currently used fluorophores. In this article we describe applications for this dye in 43 44 45 fluorometry, confocal microscopy, gel electrophoresis and flow cytometry, along with 46 47 an initial characterization of its relevant physico-chemical properties. 48 49 50 51 52 53 54 55 56 57 58 59 60 4

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1 2 3 MATERIALS AND METHODS 4 5 6 7 8 9 Methyl green solution preparation 10 11 12 We used a 2% MG stock solution obtained by chloroform extraction to remove crystal 13 14 violet impurities, according to classical methods (Kurnick and Foster 1950). Briefly, an 15 16 aqueous 4% MG (Dr. G. Grübler, Leipzig, Germany) solution was separated several 17 18 For Peer Review 19 times with chloroform until no traces of violet stain could be seen, either using a 20 21 separation funnel and discarding the lower phase (chloroform) or a test tube and 22 23 carefully recovering the upper phase (aqueous solution). 24 25 26 27 28 29 In vitro fluorometric assays 30 31 32 125 ng/mL and 10 g/mL MG solutions (1:16000 and 1:2000 from 2% stock, 33 34 35 respectively) with or without 100 g/mL calf thymus DNA were prepared in phosphate- 36 37 buffered saline (PBS). UV-visible spectra were performed with a Varian Cary 50 UV- 38 39 VIS spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Quantitation of 40 41 fluorescent emission was determined with a Cary Eclipse fluorescence 42 43 44 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) under a fixed 45 46 excitation wavelength of 633 nm recording emission from 645 - 800 nm. The data 47 48 interval was set to 1 nm, the emission slit was set to 10 nm, and the detection scan was 49 50 performed at 120 nm/min. Maximal dilutions were used whenever possible in order to 51 52 avoid internal filter effects. 53 54 55

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1 2 3 Embryo staining procedure for in situ observation 4 5 6 For in toto staining, zebrafish embryos were fixed at the desired stage with 4% 7 8 paraformaldehyde in PBS, overnight at 4º C. After washing in PBS-T (1% Triton X- 9 10 100), embryos were incubated for 3 h at room temperature to overnight at 4° C with 11 12 13 gentle agitation with the fluorophores diluted in PBS-T as follows: 2-4 g/mL MG 14 15 (1:10000 to 1:5000 from 2% stock); 0.1-0.2 g/mL propidium iodide (1:10000 to 16 17 1:5000 from 1 mg/mL stock), 1 M TO-PRO-3 (1:1000 from 1 mM stock) or 0.1-0.2 18 For Peer Review 19 20 g/mL Hoechst 33342 (1:10000 to 1:5000 from 1 mg/mL stock). Indirect 21 22 immunofluorescence was performed according to standard procedures, and MG or 23 24 Hoechst 33342 counterstaining was achieved by mixing with the secondary antibodies 25 26 27 solution and incubating overnight at 4º C. Cryosections from paraformaldehyde-fixed, 28 29 gelatin-embedded chick embryos were processed for immunofluorescence as described 30 31 (Zolessi and Arruti, 2001) and counterstained with MG and/or Hoechst 33342 by a final 32 33 incubation of 5-15 min at room temperature. Cultured cells (not shown) were treated in 34 35 a similar way. Both whole embryos and tissue sections were mounted in 70% glycerol 36 37 38 in PBS, pH 7.4. Microscopic observation and image acquisition of MG-stained tissues 39 40 was done using a spectral detection LSCM, Leica TCS-SP5 (Leica Microsystems 41 42 GmbH Wetzlar, Germany), equipped with five lasers (LED 405 nm, Argon multiple- 43 44 line 458-514 nm; HeNe 543 nm; HeNe 594 nm; HeNe 633 nm), 20x water-glycerol-oil 45 46 47 immersion (NA 0.70), 63x oil immersion (NA 1.4), and 63x water immersion (NA 1.2) 48 49 objectives. It has a resonant scanner at 800 Hz, with a maximal resolution of 1024x1024 50 51 pixels. Images were processed using open source software: FIJI-ImageJ 1.47n 52 53 (Schindelin et al. 2012) and GIMP 2.6 (GNU Image Manipulation Program). 54 55 56 57 58 59 60 6

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1 2 3 In situ quantitation of fluorescent DNA staining 4 5 6 Quantitative assessment of MG, propidium iodide and TO-PRO-3 fluorescence in situ 7 8 was performed using whole-mounted zebrafish embryos, only stained for each of these 9 10 fluorophores. Simple optical sections of selected embryonic regions (usually neural 11 12 13 retina or brain because of their high nuclear density) were obtained under different 14 15 conditions. Measurements were made during image acquisition using the LAS AF 16 17 software version 2.6.0.7266 (Leica Microsystems GmbH Wetzlar, Germany) by 18 For Peer Review 19 marking an elliptical or rectangular ROI (region of interest), depending on the shape and 20 21 22 size of the observed tissue. In all cases, we used a 63x water immersion objective (1.2 23 24 NA), simple scans (no averaging or accumulation), the pinhole set at Airy 1, a 25 26 1024x1024 scanning field and a scanner zoom of 1.7x. 27 28 29 For registering the whole emission spectrum, the xyλ scanning mode was used, with a 30 31 detection window of 5 nm and a 3 nm step, spanning a wide range in the visible 32 33 spectrum. Emission intensity at different laser excitation lines was determined by 34 35 36 performing short spectra (xy λ) of 4-7 points around the region of maximal emission for 37 38 each fluorophore (propidium iodide, 640-655 nm; TOPRO-3, 651-662 nm; MG, 664- 39 40 682 nm), and picking the maximum value in each measurement. Photobleaching was 41 42 43 assessed either by a continuous xyt acquisition or by quantitating the spectrum peak 44 45 (xy λ) at different times during continuous excitation with laser for up to 30 min. Laser 46 47 line used was 633 nm for MG and TO-PRO-3 and 488 nm for propidium iodide, in most 48 49 cases at 30% power (except when noted in figure or text). 50 51 52 53 54 55 56 57 58 59 60 7

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1 2 3 MG Staining of DNA for agarose gels 4 5 6 MG was incorporated as a 1:5000-1:10.000 dilution of the stock into a 1% agarose gel 7 8 matrix in TAE. Ethidium bromide was incorporated at 0.5 g/ml. MG and ethidium 9 10 11 bromide gels were loaded with DNA (MassRuler sm0403 from Thermo Fisher 12 13 Scientific, Waltham, for quantitative purposes; or 100 bp DNA ladder from Life 14 15 Technologies, Carlsbad, for comparative gels) or RNA (0.5-10 kb RNA ladder, 16 17 Invitrogen) molecular weight ladders at different concentrations and electrophoresed at 18 For Peer Review 19 the same time using standard methods. Images of the electrophoresed nucleic acids were 20 21 22 obtained with a G:Box (Syngene, Frederick, USA) either under epi-illumination with 23 24 red light (635 nm) and a 705M band-pass filter, with a 30-60 s exposure, or with a 25 26 standard mid-wave UV transillumination. 27 28 29 30 31 32 Cell viability assays 33 34 35 For microscopic assessment of viability, human peripheral blood mononuclear cells 36 37 (PBMCs) from healthy donors were thawed from liquid N , incubated for 30 min in 38 2 39 40 RPMI supplemented with 10% FBS and immediately prior to confocal imaging were 41 42 incubated with 0.1 volume of a mixture of 100 g/mL acridine orange, 100 g/mL 43 44 ethidium bromide and 4 g/mL MG. For flow cytometric determination of cell viability 45 46 5 47 human Raji (B-cell Burkitt's lymphoma) cells (5x10 ) purchased at ATCC (Manassas, 48 49 USA) were cultured in HEPES-buffered RPMI 1640 medium supplemented with 10% 50 51 fetal calf serum, 50 g/mL gentamicin, and 2 mM L-glutamine at 37 °C in a humidified 52 53 CO 2 atmosphere in absence or presence of Fludarabine (9-β-d-arabinosyl-2- 54 55 fluoroadenine-monophosphate) obtained from Sigma-Aldrich used at 50 or 100 µg/mL 56 57 58 (Gandhi and Plunkett 2002). After 24 hours of incubation, cells were labeled using 10 59 60 8

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1 2 3 g/mL propidium iodide and 4 g/mL MG and immediately acquired in a CyAn Flow 4 5 Cytometer (Beckman Coulter, Indianapolis, USA). Data was analyzed using Summit 6 7 8 v4.3 software from Dako Inc., USA. Markers were settled according to the fluorescent 9 10 signals detected in unstained cells, using the same acquisition parameters as in labeled 11 12 cells. 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 9

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1 2 3 RESULTS 4 5 6 7 8 9 MG absorption and emission profiles in solution 10 11 12 To initially characterize MG fluorescence properties, we performed a light absorption 13 14 spectrum of MG in a spectrophotometric assay, and determined it had three absorption 15 16 maxima at 244, 377 and 633 nm (Fig. 1a). Adding DNA to the solution did not alter this 17 18 For Peer Review 19 absorption profile. 20 21 22 Excitation of MG trough excitation at these three wavelengths in the absence of DNA, 23 24 resulted in an emission peak at 488 nm when an excitation beam of 244 nm was used, 25 26 and a smaller peak with an emission maximum at 750 nm, when a 377 nm light was 27 28 used. No fluorescence emission was detected when exciting MG in solution at 633 nm 29 30 31 (Fig. 1b). 32 33 34 We further analyzed the 633 nm absorption maximum by comparing the fluorometric 35 36 emission profile of MG alone and in the presence of DNA (Fig. 1c). Surprisingly, an 37 38 important emission peak appeared at the range of 663 - 686 nm when DNA was added, 39 40 with a maximum that is around 7.5 times higher than the corresponding curve range in 41 42 43 the absence of DNA. This peak showed a sustained decrease, reaching background 44 45 levels just after 800 nm, and indicated that MG fluorescence properties dramatically 46 47 change after binding to DNA. 48 49 50 51 52 53 54 55 56 57 58 59 60 10

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1 2 3 MG fluorescence upon nuclear staining: excitation and emission. 4 5 6 To further characterize DNA-bound MG fluorescence, we used it at diluted aqueous 7 8 solutions, unable to cause visible color staining, to label nuclei on fixed tissues. Using 9 10 whole-mounted zebrafish embryos stained with MG, we assessed the ideal excitation 11 12 13 conditions for spectral detection confocal microscopy, using laser lines 458, 476, 488, 14 15 496, 514, 543, 594 and 633 nm. As expected, maximal fluorescence intensity for MG at 16 17 the DNA-specific range was achieved at λ = 633 nm (Fig. 2a), hence this is the 18 For Peer Review 19 excitation wavelength we used in subsequent microscopy experiments. With the other 20 21 22 laser excitation lines, MG far-red fluorescent signal was very close to background. 23 24 Remarkably, TO-PRO-3 gave a nearly identical excitation curve, also with a maximum 25 26 at 633 nm, while propidium iodide showed a wider range of excitation, with two 27 28 maximal peaks at 488 and 514 nm and signal significantly above background between 29 30 476 and 543 nm (Fig. 2a). 31 32 33 Using the spectral quantitative mode in the LSCM (LAS-AF acquisition software), we 34 35 36 also measured the emission spectra of these three red/far-red emitting fluorophores. 37 38 Similar to what was seen in solution, maximal signal for MG was achieved at 677 nm, 39 40 rather far from the excitation wavelength at 633 nm, while for TO-PRO-3 it was 41 42 relatively closer, at 649 nm (Fig. 2b). Propidium iodide fluorescence emission was 43 44 45 measured by exciting at its 488 nm peak to give a cleaner spectrum, and in these 46 47 conditions maximal signal was at 609 nm (Fig. 2b). As the emission spectra were 48 49 clearly different in shape, we compared sharpness of the peaks by measuring their width 50 51 at 50% of the maximal intensity. TO-PRO-3 gave the sharpest spectrum, with a 50% 52 53 54 emission band of 35 nm (636-671 nm), followed by MG with 67 nm (649-716 nm) and 55 56 propidium iodide with 87 nm (584-671 nm) (Fig. 2b). 57 58 59 60 11

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1 2 3 MG fluorescence upon nuclear staining: photobleaching. 4 5 6 Photobleaching is another usual inconvenient of many fluorophores, particularly when 7 8 high resolution confocal images of thick specimens is desired, requiring long exposure 9 10 times. We assessed sensitivity of MG and the other fluorophores to photobleaching by 11 12 13 continuous excitation with the appropriate laser at 30% power. While in these 14 15 conditions TO-PRO-3 signal decayed to undetectable in less than 2 minutes when 16 17 exciting with the 633 nm laser, MG signal remained extremely stable for a very long 18 For Peer Review 19 time, with a loss of around only 1% in 30 minutes (Fig. 2c). Using a 100% percent laser 20 21 22 power gave very similar results for MG photobleaching (not shown). Propidium iodide 23 24 also proved to be rather stable when excited at 488 nm (albeit much less than MG), 25 26 losing around 10% of its fluorescence signal in 4-5 minutes (Fig. 2c). 27 28 29 30 31 32 MG as an ideal fluorescent nuclear stain for embryonic tissues 33 34 35 We assayed MG ability to be used as a nuclear stain for whole-mounted and sectioned 36 37 embryonic tissues, using zebrafish whole embryos and chick embryo cryosections (Fig. 38 39 3). Remarkably, whole body staining of zebrafish embryos was achieved within 3 hours 40 41 42 of incubation. Confocal sections of the zebrafish retina and olfactory pit, as well as 43 44 chick neural plate region, revealed staining of DNA with very good sub-nuclear 45 46 resolution (Fig. 3). This label was stable and it did not interfere with other fluorophores, 47 48 antibodies or phalloidin actin labeling (Fig. 3). Furthermore, we did not find 49 50 51 interference with other DNA stains such as Hoechst 33342, a minor groove-interacting 52 53 molecule. Figure 4 shows two series of images taken from chick embryo cryosections at 54 55 neurula stage, doubly labeled with Hoechst 33342 and MG. When tissues were 56 57 massively irradiated for zoomed images using the 63x water immersion objective (1.2 58 59 60 12

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1 2 3 NA), MG labeling did not exhibit a noticeable decay in fluorescence emission, as 4 5 expected, allowing us to acquire 0.2 m spaced z-stacks for 3D reconstruction in both, 6 7 8 zebrafish whole embryos and chick embryo sections (see an example in Online 9 10 Resource 1). 11 12 13 14 15 16 MG as a staining procedure specific for DNA in agarose gels 17 18 For Peer Review 19 As MG can fluorescently label DNA in vitro , we examined its suitability for staining 20 21 electrophoresed DNA in agarose gels. We analyzed the ability of MG to stain different 22 23 amounts of DNA fragments of different sizes using a quantitated commercial DNA 24 25 ladder (Fig. 5a). Interestingly, MG proved to be able to allow for the detection of at 26 27 28 least 4.8 ng of a 1.5 kb DNA fragment (Figure 5a, lane 2, arrowhead) under red (635 nm) 29 30 epi-illumination. As the most commonly used stain for DNA in gels is ethidium 31 32 bromide, we also decided to compare MG with this stain for the labeling of 33 34 electrophoresed DNA and RNA. Separate gels with either stain were identically loaded 35 36 37 with samples of commercial DNA and RNA ladder. Interestingly, while ethidium 38 39 bromide labeled, as it is well known, both nucleic acids with similar intensities, MG 40 41 was only able to efficiently detect DNA (Fig. 5b). An extremely weak RNA label could 42 43 be detected, indicating that even if MG might be able to show some RNA binding, its 44 45 46 affinity for RNA must be several times lower than that for DNA. 47 48 49 50 51 52 MG as a cell viability stain in fresh and cultured cells 53 54 55 To assess the ability of MG to act as a vital stain, we labeled live human PBMCs from 56 57 healthy donors and found it to stain only a small fraction of the cells (not shown). This 58 59 60 13

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1 2 3 observation made us wonder if viable cells could be actively excluding MG, and if 4 5 labeled cells could actually be dead or dying. Some DNA intercalating molecules, such 6 7 as propidium iodide and ethidium bromide show this property, and are hence very 8 9 10 commonly used to assess cell viability. When comparing MG with ethidium bromide on 11 12 these living cells, we found a 1:1 correlation on the number of labeled cells (Fig. 6a), 13 14 indicating that MG can also be used to assess cell viability. In contrast, the cell 15 16 permeant intercalating stain acridine orange labeled the full cell population under the 17 18 For Peer Review 19 same conditions (Fig. 6a). 20 21 22 Finally, we evaluated the performance of MG as a direct measure of cell viability by 23 24 flow cytometry. We chose to work with Raji cells, a lymphoblastic-like B line of human 25 26 origin. As shown in the upper charts of Figure 6b, forward versus size gating of viable 27 28 or dead cells (R1 or R2) correlated with low and high percentage of positive cells 29 30 respectively, for both MG and the well-known viability stain propidium iodide. This 31 32 33 shows a correlation in the percentages of cell death and cell viability between forward 34 35 versus size cytometric analysis and MG positive and negative stain, respectively. In 36 37 addition, when treated with the cytotoxic purine analog Fludarabine, usually used in 38 39 cancer chemotherapy, MG stained Raji cells in identical percentages as compared to 40 41 42 propidium iodide. As expected, this combined staining protocol resulted in double 43 + + 44 positive PI MG cells, as depicted in the representative dot plots presented in the lower 45 46 charts of Figure 6b. This result confirms that MG is only able to label dead cells nuclei, 47 48 in a similar manner as propidium iodide, and shows that it can be used in fluorescent- 49 50 based techniques to discriminate dead or damaged cells by positive staining. 51 52 53

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1 2 3 DISCUSSION 4 5 6 7 8 9 Until now, tissue staining protocols used MG in concentrations several orders of 10 11 magnitude larger than shown here (usual histological stain uses around 0.1-0.5% 12 13 solutions). Under such conditions, MG also acts as an excellent quencher of unspecific 14 15 16 staining of nuclei and this is why it has been used as a counterstain of fluorescently 17 18 labeled nucleoli For(Pollack et al.Peer 1982). It would Review not be surprising that interactions of the 19 20 sort described for pyronin Y by Kapuscinski and Darzynkiewicz (Kapuscinski and 21 22 Darzynkiewicz 1987) would explain the fact that a molecule able to act as a fluorescent 23 24 25 stain could switch its behavior to that of a quencher in a concentration-dependent 26 27 manner. This fact, together with the lack of a generalized availability of spectral 28 29 confocal microscopes, probably hindered MG's notable properties as a fluorescent stain. 30 31 We could find only one report in the literature on the use of MG as a fluorescent DNA 32 33 stain on histological preparations (Itoh et al. 2003). In this case, however, a high 34 35 36 concentration of MG was used to generate the usual green stain visible in bright field 37 38 microscopy, and the fluorescence excitation-emission wavelengths were coincident with 39 40 those of orange-red fluorophores such as TRITC, hence very different to the 41 42 advantageous properties reported in the present work. 43 44 45 We have shown here MG's excellent qualification as a general purpose DNA staining 46 47 48 for fluorescence microscopy with sub-nuclear resolution, and high emission levels even 49 50 after being irradiated during extended periods of time. The staining procedure is 51 52 extremely simple, not to mention its low cost and shelf-life stability (the stock solution 53 54 is stable for months at room temperature). After its comparison with two other 55 56 extensively used red fluorescent stains for nuclei, propidium iodide and TO-PRO-3, we 57 58 59 60 15

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1 2 3 found a much superior performance for MG. Like TO-PRO-3, MG shows a maximal 4 5 excitation at 633 nm and a relatively narrow emission peak at the far-red wavelength 6 7 range, while propidium iodide is excited by a wide range of shorter wavelengths, 8 9 10 especially 488 and 514 nm, while emitting in an extremely wide range in the visible 11 12 spectrum. Not only its excitation, but also its emission range, are coincident with that of 13 14 several other common-use fluorophores such as FITC or TRITC (or more recently 15 16 developed similar ones like the corresponding Alexas), making it a bad option for multi- 17 18 For Peer Review 19 color fluorescence imaging. With MG (or TO-PRO-3), however, this problem is almost 20 21 inexistent as they do not or very little overlap with excitation or emission spectra of 22 23 common-use blue, green or orange-red fluorophores. Indeed, MG very little overlaps 24 25 with TRITC or Alexa 594, while TO-PRO-3, with an emission spectrum relatively blue- 26 27 shifted, even if still little overlapping with TRITC, will have a larger overlap with Alexa 28 29 30 594 leading to the possibility of bleed-through. The Stokes shift is also much better for 31 32 MG (44 nm) than for TO-PRO-3 (16 nm). Finally, one major problem with the very 33 34 good DNA stain TO-PRO-3 is that it bleaches relatively quickly when excited, even for 35 36 short periods of time. MG showed an astonishing stability of its fluorescence upon 37 38 39 continuous excitation, even at the highest laser power. A major advantage relies in the 40 41 fact that MG does not fluoresce when irradiated at 633 nm on its own, but increases 42 43 several times its emission intensity when bound to DNA, making it suitable for being 44 45 added to mounting media as it is unnecessary to wash out the remaining MG from the 46 47 samples. 48 49 50 These characteristics position MG as a central molecule for routine DNA staining in 51 52 53 confocal microscopy protocols for whole embryos, as well as tissue sections or cultured 54 55 cells. The recent development of different techniques that allow for the detailed 56 57 observation of whole samples of tissue, such as light sheet microscopy (Amat and 58 59 60 16

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1 2 3 Keller 2013) or the CLARITY method for tissue diaphanization (Chung et al. 2013), 4 5 can be greatly benefited from a very stable stain like this. The fact that both excitation 6 7 and emission of MG are in the long-wavelength region of the spectrum also facilitates 8 9 10 light penetration in both directions, with the expected generation of better quality 11 12 nuclear images from thick specimens than those obtained with the more commonly used 13 14 blue-emitting fluorophores (Lenz 1999). In our hands, MG did not interfere with 15 16 staining with other fluorescent probes, such as small molecules, antibodies or 17 18 For Peer Review 19 fluorescent proteins. Regarding this, in our method MG is diluted in physiological pH 20 21 buffers, with or without proteins or nonionic detergents, while the classical histological 22 23 technique implies using an acetate buffer at pH 4.2, which would be incompatible with 24 25 antibody co-incubation. 26 27 28 Furthermore the ability of MG to stain DNA in agarose gels combined with its spectral 29 30 characteristics, together with the fact that its interaction with DNA is non-intercalating 31 32 33 but electrostatic and that its sensitivity is similar to that of ethidium bromide - not to 34 35 mention its lower market value when compared to usual commercial stains - may put 36 37 MG on the bench of every molecular biology lab. It is remarkable that we have assayed 38 39 it under epi-illumination and found it to be a DNA stain at least as efficient as ethidium 40 41 42 bromide in terms of detection limits. It seems probable that under strong trans- 43 44 illumination conditions - such as those achieved by led arrays, it may show even better 45 46 performance than we have shown here. Even if it has been classically shown that MG 47 48 only labels DNA when used as a colored stain on cells and it preferentially binds 49 50 double-stranded DNA over RNA (see for example Kurnick and Foster 1950; Krey and 51 52 53 Hahn 1975), and that there was no evident cytoplasmic signal in the fluorescent MG 54 55 staining of cells shown in the present work, we decided to confirm that MG 56 57 preferentially labels DNA also as a fluorescent stain. By electrophoresis gel staining, we 58 59 60 17

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1 2 3 showed here that MG has a very strong preference for DNA above RNA labeling. This 4 5 observation also makes it a potentially very useful tool to differentiate between these 6 7 two nucleic acids in biological samples, for example in combination with the unspecific 8 9 10 compound ethidium bromide. 11 12 13 Finally, we have demonstrated the suitability of MG to act as a reliable cell viability 14 15 probe for both microscopy and flow cytometry techniques. Live peripheral PBMC 16 17 labeling evaluated by fluorescence microscopy revealed that only a small fraction of 18 For Peer Review 19 cells stained for MG, a fraction which interestingly resulted to coincide with cells 20 21 22 positive for the well-established viability staining with ethidium bromide (Kasibhatla et 23 24 al. 2006). Furthermore, we evaluated MG staining by flow cytometry in fresh Raji cells 25 26 and in induced cell-death experiments, using the cytotoxic purine analog Fludarabine. 27 28 We found that discrimination of viable and dead cells by forward versus size scattering 29 30 analysis, which represents a vastly used method for viability determination (Liegler et al. 31 32 33 1995), matched with negative MG staining of viable cells and positive MG staining of 34 35 dead cells. Importantly, the percentages of cell death determined by MG staining were 36 37 highly similar to those obtained by propidium iodide staining. Finally, Fludarabine- 38 39 induced cell death showed a similar decrease in cultured cells viability, as evaluated by 40 41 + + 42 percentages of MG cells, percentages of PI cells, and percentages of cells obtained in 43 44 forward versus size scattering analysis. Quantification of all three parameters revealed 45 46 near identical results for both compounds, where dead cells were always double positive. 47 48 49 Altogether, data presented here show that MG can be used as a general purpose 50 51 fluorescent DNA label, with excellent properties regarding excitation-emission 52 53 54 wavelengths, fluorescence intensity and stability, product cost and shelf-life. We have 55 56 also demonstrated that it can be extremely useful for multiple cell biology-related 57 58 applications, and we foresee that there are many more possible, such as DNA 59 60 18

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1 2 3 quantitation in solution, in gels or in situ , the determination of cell cycle parameters by 4 5 flow cytometry, and others. The facts that MG is an old-known compound, with a very 6 7 low commercial cost, and patent-free, make it easily accessible to any research or 8 9 10 clinical laboratory, anywhere in the world. 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 19

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1 2 3 ACKNOWLEDGEMENTS 4 5 6 The authors would like to thank Dr. Cristina Arruti and Dr. Pablo Oppezzo for 7 8 facilitating the use of reagents and lab installations; the Cell Biology Unit at the Institut 9 10 Pasteur Montevideo for access to the confocal microscope and flow cytometer; Dr. 11 12 13 Mario Señorale for access to the G:Box. Partial funding was from Comisión Sectorial 14 15 de Investigación Científica (CSIC)-Universidad de la República and PEDECIBA, 16 17 Uruguay. 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 20

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1 2 3 REFERENCES 4 5 6 7 Amat F, Keller PJ (2013) Towards comprehensive cell lineage reconstructions in 8 9 complex organisms using light-sheet microscopy. Dev Growth Differ 55 10 11 12 (4):563-578 13 14 Beumer TL, Veenstra GJ, Hage WJ, Destrée OH (1995) Whole-mount 15 16 immunohistochemistry on Xenopus embryos using far-red fluorescent dyes. 17 18 Trends inFor genetics : TIGPeer 11:9 Review 19 20 Chung K, Wallace J, Kim S-Y, Kalyanasundaram S, Andalman AS, Davidson TJ, 21 22 23 Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, 24 25 Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K (2013) Structural and 26 27 molecular interrogation of intact biological systems. Nature 497:332-337 28 29 De Petrocellis B, Parisi E (1973) Deoxyribonuclease in sea urchin embryos. 30 31 32 Comparison of the activity present in developing embryos, in nuclei, and in 33 34 mitochondria. Experimental cell research 79:53-62 35 36 Gandhi V, Plunkett W (2002) Cellular and Clinical Pharmacology of Fludarabine. 37 38 Clinical Pharmacokinetics 41 (2):93-103 39 40 Høyer PE, Lyon H, Jakobsen P, Andersen AP (1986) Standardized methyl green- 41 42 43 pyronin Y procedures using pure dyes. The Histochemical journal 18:90-94 44 45 Itoh J, Umemura S, Hasegawa H, Yasuda M, Takekoshi S, Osamura YR, Watanabe K 46 47 (2003) Simultaneous Detection of DAB and Methyl Green Signals on Apoptotic 48 49 Nuclei by Confocal Laser Scanning Microscopy. Acta Histochemica Et 50 51 52 Cytochemica 36:367-376 53 54 55 56 57 58 59 60 21

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1 2 3 Kapuscinski J (1995) DAPI: a DNA-specific fluorescent probe. Biotechnic & 4 5 histochemistry : official publication of the Biological Stain Commission 70:220- 6 7 233 8 9 10 Kapuscinski J, Darzynkiewicz Z (1987) Interactions of pyronin Y(G) with nucleic acids. 11 12 Cytometry 8:129-137 13 14 Kasibhatla S, Amarante-Mendes GP, Finucane D, Brunner T, Bossy-Wetzel E, Green 15 16 DR (2006) Acridine Orange/Ethidium Bromide (AO/EB) Staining to Detect 17 18 For Peer Review 19 Apoptosis. Cold Spring Harbor Protocols 2006 (3):pdb.prot4493 20 21 Kim SK, Nordén B (1993) Methyl green. A DNA major-groove binding drug. FEBS 22 23 letters 315:61-64 24 25 Klonisch T, Wark L, Hombach-Klonisch S, Mai S (2010) Nuclear imaging in three 26 27 dimensions: a unique tool in cancer research. Annals of anatomy = 28 29 30 Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft 31 32 192:292-301 33 34 Krey AK, Hahn FE (1975) Studies on the Methyl Green-DNA complex and its 35 36 dissociation by drugs. Biochemistry 14:5061-5067 37 38 39 Kurnick NB (1952) The basis for the specificity of methyl green staining. Experimental 40 41 Cell Research 3:649-651 42 43 Kurnick NB, Foster M (1950) Methyl green. III. Reaction with desoxyribonucleic acid, 44 45 stoichiometry, and behavior of the reaction product. The Journal of general 46 47 physiology 34:147-159 48 49 50 Kurnick NB, Sandeen G (1960) Acid desoxyribonuclease assay by the methyl green 51 52 method. Biochimica et biophysica acta 39:226-231 53 54 Latt SA, Stetten G (1976) Spectral studies on 33258 Hoechst and related 55 56 bisbenzimidazole dyes useful for fluorescent detection of deoxyribonucleic acid 57 58 59 60 22

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1 2 3 synthesis. The journal of histochemistry and cytochemistry : official journal of 4 5 the Histochemistry Society 24:24-33 6 7 Lenz P (1999) Fluorescence measurement in thick tissue layers by linear or nonlinear 8 9 10 long-wavelength excitation. Appl Opt 38 (16):3662-3669 11 12 Li B, Wu Y, Gao X-M (2003) Pyronin Y as a fluorescent stain for paraffin sections. The 13 14 Histochemical journal 34:299-303 15 16 Liegler TJ, Hyun W, Yen TS, Stites DP (1995) Detection and quantification of live, 17 18 For Peer Review 19 apoptotic, and necrotic human peripheral lymphocytes by single-laser flow 20 21 cytometry. Clin Diagn Lab Immunol 2 (3):369-376 22 23 Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, 24 25 Leung W-Y (1999) Alexa Dyes, a Series of New Fluorescent Dyes that Yield 26 27 Exceptionally Bright, Photostable Conjugates. Journal of Histochemistry & 28 29 30 Cytochemistry 47:1179-1188 31 32 Pollack A, Prudhomme DL, Greenstein DB, Irvin GL, Claflin AJ, Block NL (1982) 33 34 Flow cytometric analysis of RNA content in different cell populations using 35 36 pyronin Y and methyl green. Cytometry 3:28-35 37 38 39 Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch 40 41 S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White DJ, Hartenstein V, 42 43 Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for 44 45 biological-image analysis. Nature methods 9:676-682 46 47 Terai T, Nagano T (2013) Small-molecule fluorophores and fluorescent probes for 48 49 50 bioimaging. Pflügers Archiv : European journal of physiology 465:347-359 51 52 Van Hooijdonk CA, Glade CP, Van Erp PE (1994) TO-PRO-3 iodide: a novel HeNe 53 54 laser-excitable DNA stain as an alternative for propidium iodide in 55 56 multiparameter flow cytometry. Cytometry 17:185-189 57 58 59 60 23

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1 2 3 Zolessi FR, Arruti C (2001) Apical accumulation of MARCKS in neural plate cells 4 5 during neurulation in the chick embryo. BMC Dev Biol 1:7 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37 38 39 40 41 42 43 44 45 46 47 48

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1 2 3 FIGURE CAPTIONS 4 5 6 7 Fig. 1 Spectral properties of methyl green in solution. a Absorption spectra of MG (10 8 9 g/mL), both free and in the presence of DNA, displayed maxima at 244, 377 and 633 10 11 12 nm. In the presence of DNA, the DNA 280 nm absorption peak was added, plus a 13 14 protein contamination peak at 260 nm. b Fluorescent emission of free MG (125 ng/mL) 15 16 when 244, 377 and 633 nm excitation wavelengths were applied. Excitation at 244 nm 17 18 elicited an emissionFor peak at Peer488 nm; excitation Review at 377 nm elicited an emission peak at 19 20 21 750 nm; excitation at 633 nm elicited no detectable emission peaks. c Excitation of MG 22 23 (10 g/mL) at 633 nm results in an emission peak with a maximum at 650 nm in the 24 25 presence of DNA 26 27 28 29 30 31 Fig. 2 Quantitative assessment of methyl green properties as a fluorophore for laser 32 33 excitation microscopy. a Comparative fluorescence response of MG, TO-PRO-3 and 34 35 36 propidium iodide (PI), measured at the maximum emission peak for each fluorophore, 37 38 upon the excitation with different laser lines with a spectral detection laser scanning 39 40 confocal microscope. b Emission intensity spectra of propidium iodide (PI), TO-PRO-3 41 42 and MG, exciting with their respective ideal laser lines, as determined in a. c Temporal 43 44 profile of fluorescence emission decay for propidium iodide (PI), TO-PRO-3 and MG 45 46 47 after continuous excitation with their ideal laser lines (30% power). The inset shows a 48 49 comparison of the fluorescence emission decay only for TOPRO-3 and MG after a 50 51 prolonged time of continuous excitation (30 min), measuring at 5 min intervals 52 53 54 55 56 57 58 59 60 25

Histochemistry and Cell Biology Page 26 of 65

1 2 3 Fig. 3 Examples of embryonic tissues stained with methyl green and different 4 5 fluorescent markers. a Confocal section through the retina of a whole-mounted 60 hpf 6 7 zebrafish embryo labeled with MG and TRITC-conjugated Phalloidin to highlight 8 9 10 regions enriched in actin filaments. MG clearly demarcates all nuclei and even allows 11 12 for the identification of different cell types based on nuclear size and shape, as well as 13 14 chromatin condensation. The arrowhead points to a pyknotic nucleus. am, amacrine 15 16 cells in the inner nuclear layer; pr; photoreceptors; rgc, retinal ganglion cells; rpe, 17 18 For Peer Review 19 retinal pigmentary epithelium. b Maximum intensity projection of 10 confocal slices 20 21 obtained from a chick embryo cryosection, labeled with MG and TRITC-Phalloidin. 22 23 The image shows the region of the neural plate-epidermal border. Several mitotic cells 24 25 are clearly evidenced by the very strong MG labeling of their chromosomes 26 27 (arrowheads). ep, presumptive epidermis; np, neural plate. c Maximum intensity 28 29 30 projection of 3 confocal slices from a whole-mounted 48 hpf zebrafish embryo labeled 31 32 with MG, TRITC-Phalloidin and an antibody to acetylated tubulin (highlighting cilia). 33 34 The high magnification image shows the olfactory pit, where MG very clearly labels 35 36 subnuclear chromatin structure. Scale bars: a-b, 20 m; c, 5 m 37 38 39

40 41 42 Fig. 4 High magnification confocal sections of chick embryonic tissue double-labeled 43 44 45 with MG and Hoechst 33342. a Series of selected optical sections through a nucleus 46 47 from an ectodermal cell of a neurula-stage chick embryo cryosection. Comparison of 48 49 MG and Hoechst staining of the same nuclei, showing nuclear and sub-nuclear structure. 50 51 The arrowheads point to condensed chromatin structures highlighted by both 52 53 54 compounds, but with different relative levels of intensity. b Maximum intensity 55 56 projection of a confocal stack from a chick neurula cryosection, showing interphasic 57 58 and mitotic endodermal cells. Mitotic chromosome morphology is revealed at very 59 60 26

Page 27 of 65 Histochemistry and Cell Biology

1 2 3 similar resolution and staining pattern for both compounds. See a 3D reconstruction in 4 5 Online Resource 1. Scale bars: 5 m 6 7 8 9 10 11 Fig. 5 Methyl green as an efficient and specific fluorescent stain for DNA gel 12 13 14 electrophoresis. a DNA in an agarose gel electrophoresis was stained with a solution of 15 16 MG as described in the text. Gel lanes were loaded with the indicated volumes of DNA 17 18 ladder (at 103 ng/Forl). The limitPeer of detection Review was determined as the 4.8 ng band of the 19 20 second lane (arrowhead). b Comparison of DNA and RNA labeling on gels with MG 21 22 23 and ethidium bromide. Ethidium bromide (left) and MG (right) were incorporated into 24 25 agarose gels that were loaded with the indicated amounts of DNA or RNA ladder. After 26 27 electrophoresis, label was evidenced using the appropriate illumination. Negative 28 29 images of the fluorescence on gels are shown in a and b. 30 31 32 33 34 35 Fig. 6 Methyl green as a cell viability stain for fluorescence microscopy and flow 36 37 cytometry. a Confocal sections of live PBMCs stained with acridine orange (AO), 38 39 40 methyl green (MG) and ethidium bromide (EB). Lower right panel shows EB/MG 41 42 images overlay, evidencing complete colocalization. Scale bar: 5 m. b Flow 43 44 cytometric quantitation of cell viability comparing size/granularity, propidium iodide 45 46 47 (PI) and MG staining in cultured Raji cells. Cells from the human B cell line Raji

48 5 49 (5x10 ) were cultured in the absence or presence of Fludarabine for 24 h at the indicated 50 51 concentrations. After the incubations, cells were stained for propidium iodide (PI) and 52 53 methyl green (MG) labeling and immediately acquired in a flow cytometer. Shown are 54 55 representative dot plots of flow cytometry analyses depicting forward versus size 56 57 58 scattering, PI or MG histograms, or PI and MG double staining. Percentages of positive 59 60 27

Histochemistry and Cell Biology Page 28 of 65

1 2 3 cells are indicated (n=3 independent experiments). Forward versus size scattering 4 5 comparative analysis of MG and propidium iodide indicates that MG only stained dead 6 7 cells 8 9 10

11 12 13 Online Resource 1 (Movie) 14 15 16 3D reconstruction of a mitotic cell from chick embryo stained with MG, Hoechst 33342 17 18 For Peer Review 19 and TRITC-Phalloidin showing that MG provides very good levels of image resolution 20 21 at x/y and z axes. Green, MG; blue, Hoechst 33342 (shown merged with the green 22 23 channel); red, TRITC-Phalloidin 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 28

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 1 Spectral properties of methyl green in solution. a Absorption spectra of MG (10 µg/mL), both free and in the presence of DNA, displayed maxima at 244, 377 and 633 nm. In the presence of DNA, the DNA 280 48 nm absorption pea k was added, plus a protein contamination peak at 260 nm. b Fluorescent emission of free 49 MG (125 ng/mL) when 244, 377 and 633 nm excitation wavelengths were applied. Excitation at 244 nm 50 elicited an emission peak at 488 nm; excitation at 377 nm elicited an emission peak at 750 nm; excitation 51 at 633 nm elicited no detectable emission peaks. c Excitation of MG (10 µg/mL) at 633 nm results in an 52 emission peak with a maximum at 650 nm in the presence of DNA 53 279x548mm (300 x 300 DPI) 54 55 56 57 58 59 60 Histochemistry and Cell Biology Page 58 of 65

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 2 Quantitative assessment of methyl green properties as a fluorophore for laser excitation microscopy. a Comparative fluorescence response of MG, TO-PRO-3 and propidium iodide (PI), measured at the maximum 48 emission peak for each fluorophore, upon the excitation with different laser lines with a spectral detection 49 laser scanning confocal microscope. b Emission intensity spectra of propidium iodide (PI), TO-PRO-3 and 50 MG, exciting with their respective ideal laser lines, as determined in a. c Temporal profile of fluorescence 51 emission decay for propidium iodide (PI), TO-PRO-3 and MG after continuous excitation with their ideal laser 52 lines (30% power). The inset shows a comparison of the fluorescence emission decay only for TOPRO-3 and 53 MG after a prolonged time of continuous excitation (30 min), measuring at 5 min intervals 54 285x731mm (300 x 300 DPI) 55 56 57 58 59 60 Page 59 of 65 Histochemistry and Cell Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 3 Examples of embryonic tissues stained with methyl green and different fluorescent markers. a Confocal section through the retina of a whole-mounted 60 hpf zebrafish embryo labeled with MG and 48 TRITC-conjugated Phalloidin to highlight regions enriched in actin filaments. MG clearly demarcates all nuclei 49 and even allows for the identification of different cell types based on nuclear size and shape, as well as 50 chromatin condensation. The arrowhead points to a pyknotic nucleus. am, amacrine cells in the inner 51 nuclear layer; pr; photoreceptors; rgc, retinal ganglion cells; rpe, retinal pigmentary epithelium. b Maximum 52 intensity projection of 10 confocal slices obtained from a chick embryo cryosection, labeled with MG and 53 TRITC-Phalloidin. The image shows the region of the neural plate-epidermal border. Several mitotic cells are 54 clearly evidenced by the very strong MG labeling of their chromosomes (arrowheads). ep, presumptive 55 epidermis; np, neural plate. c Maximum intensity projection of 3 confocal slices from a whole-mounted 48 hpf zebrafish embryo labeled with MG, TRITC-Phalloidin and an antibody to acetylated tubulin (highlighting 56 cilia). The high magnification image shows the olfactory pit, where MG very clearly labels subnuclear 57 chromatin structure. Scale bars: a-b, 20 µm; c, 5 µm 58 59 60 Page 61 of 65 Histochemistry and Cell Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Fig. 4 High magnification confocal sections of chick embryonic tissue double-labeled with MG and Hoechst 44 33342. a Series of selected optical sections through a nucleus from an ectodermal cell of a neurula-stage 45 chick embryo cryosection. Comparison of MG and Hoechst staining of the same nuclei, showing nuclear and 46 sub-nuclear structure. The arrowheads point to condensed chromatin structures highlighted by both 47 compounds, but with different relative levels of intensity. b Maximum intensity pro jection of a confocal stack 48 from a chick neurula cryosection, showing interphasic and mitotic endodermal cells. Mitotic chromosome 49 morphology is revealed at very similar resolution and staining pattern for both compounds. See a 3D 50 reconstruction in Online Resource 1. Scale bars: 5 µm 184x202mm (300 x 300 DPI) 51 52 53 54 55 56 57 58 59 60 Histochemistry and Cell Biology Page 62 of 65

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Methyl green as an efficient and specific fluorescent stain for DNA gel electrophoresis. a DNA in an agarose gel electrophoresis was stained with a solution of MG as described in the text. Gel lanes were loaded with 48 the indicated volumes of DNA ladder (at 103 ng/µl). The limit of detection was determined as the 4.8 ng 49 band of the second lane (arrowhead). b Comparison of DNA and RNA labeling on gels with MG and ethidium 50 bromide. Ethidium bromide (left) and MG (right) were incorporated into agarose gels that were loaded with 51 the indicated amounts of DNA or RNA ladder. After electrophoresis, label was evidenced using the 52 appropriate illumination. Negative images of the fluorescence on gels are shown in a and b. 53 101x124mm (300 x 300 DPI) 54 55 56 57 58 59 60 Page 63 of 65 Histochemistry and Cell Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 6 Methyl green as a cell viability stain for fluorescence microscopy and flow cytometry. a Confocal sections of live PBMCs stained with acridine orange (AO), methyl green (MG) and ethidium bromide (EB). 48 Lower right panel s hows EB/MG images overlay, evidencing complete colocalization. Scale bar: 5 µm. b Flow 49 cytometric quantitation of cell viability comparing size/granularity, propidium iodide (PI) and MG staining in 50 cultured Raji cells. Cells from the human B cell line Raji (5x10 5) were cultured in the absence or presence of 51 Fludarabine for 24 h at the indicated concentrations. After the incubations, cells were stained for propidium 52 iodide (PI) and methyl green (MG) labeling and immediately acquired in a flow cytometer. Shown are 53 representative dot plots of flow cytometry analyses depicting forward versus size scattering, PI or MG 54 histograms, or PI and MG double staining. Percentages of positive cells are indicated (n=3 independent 55 experiments). Forward versus size scattering comparative analysis of MG and propidium iodide indicates that MG only stained dead cells 56 230x601mm (300 x 300 DPI) 57 58 59 60 Page 65 of 65 Histochemistry and Cell Biology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Fig. 4 High magnification confocal sections of chick embryonic tissue double-labeled with MG and Hoechst 33342. a Series of selected optical sections through a nucleus from an ectodermal cell of a neurula-stage 48 chick embryo cryosection. Comparison of MG and Hoechst staining of the same nuclei, showing nuclear and 49 sub-nuclear structure. The arrowheads point to condensed chromatin structures highlighted by both 50 compounds, but with different relative levels of intensity. b Maximum intensity pro jection of a confocal stack 51 from a chick neurula cryosection, showing interphasic and mitotic endodermal cells. Mitotic chromosome 52 morphology is revealed at very similar resolution and staining pattern for both compounds. See a 3D 53 reconstruction in Online Resource 1. Scale bars: 5 µm 54 209x289mm (300 x 300 DPI) 55 56 57 58 59 60 Histochemistry and Cell Biology Page 68 of 65