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Histochemistry and Cell Biology Draft Manuscript for Review 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 Date Submitted by the Author: n/a 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 Note: The following files were submitted by the author for peer review, but cannot be converted to PDF. You must view these files (e.g. movies) online. ESM_1.mpg 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 Histochemistry and Cell Biology Page 2 of 65 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 Page 3 of 65 Histochemistry and Cell Biology 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 Histochemistry and Cell Biology Page 4 of 65 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 Page 5 of 65 Histochemistry and Cell Biology 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 µg/mL MG solutions (1:16000 and 1:2000 from 2% stock, 33 34 35 respectively) with or without 100 µg/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 56 57 58 59 60 5 Histochemistry and Cell Biology Page 6 of 65 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.

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