Acta Histochem. Cytochem. 36 (4): 361–366, 2003 Prof. K Watanabe Memorial Article Methyl Green Staining and DNA Strands In Vitro: High Affinity of Methyl Green Dye to Cytosine and Guanine Shinobu Umemura1, Johbu Itoh2, Susumu Takekoshi1, Hideaki Hasegawa2, Masanori Yasuda1, R. Yoshiyuki Osamura1 and Keiichi Watanabe1 1Department of Pathology, 2Laboratory of Structure and Function Research, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259–1193, Japan Received May 23, 2003; accepted June 25, 2003 We occasionally encounter decreased more strongly than low concentrated staining of methyl green (MG) in heated SSC or distilled water. Denatured and/or sections for antigen retrieval. To exam- DNase treated DNA showed decreased ine the relation between MG staining staining. It was especially noteworthy and conditions of DNA strands, DNA in that oligonucleotide DNA strands of poly various conditions were blotted on the C and poly G strands were positively transfer membrane and stained. It was stained, but very faint MG staining was found that staining intensity of MG was also observed for poly A while no stain- changed due to structural alterations of ing was noted for poly T. In conclusion, DNA strands. Double stranded DNA in we presented that MG staining intensity various solutions, denatured single- was altered by the conditions of DNA in stranded DNA, DNase treated samples, vitro. Intense MG staining for basic acids and short length single-stranded oligo- with three hydrogen bonds, cytosine and nucleotides were examined. DNA diluted guanine, suggest a possible mechanism in high concentrated SSC was stained for MG staining. Key words: methyl green, DNA strand, cytosine and guanine, hydrogen bond, dot blotting I. Introduction ever, stained as strongly as RNA by pyronin [5, 6]. Sub- sequent studies showed that the affinity of MG and pyronin Methyl green (MG) staining is widely utilized for to DNA or RNA were not determined by chemical differ- nuclear staining, ever since Brachet reported that Pappen- ences, but depended on the conditions of gene strands [11] heim’s (1899) mixture of methyl green was useful for stain- or, more simply, by the staining speeds by both dyes [9]. We ing chromatin DNA [2, 3]. MG is especially useful for therefore had a hypothesis that the decreased MG staining nuclear counterstaining in immunohistochemistry, because is caused by conformational changes of DNA caused by the color of MG itself is of low contrast compared to heating. Many histochemical stainings are of practical use, Hematoxyrin, and can highlight positive nuclear staining, but the principles and mechanisms of staining have not especially in black-and-white photographs. However, we oc- always been well documented. Especially when using paraf- casionally encounter decreased or no MG staining in heat- fin sections taken from in vivo tissue, we cannot observe induced antigen retrieved sections [7], the reason for which how MG dye binds to some of the specific molecules such is not fully understood. as DNA strands, or how it is altered by heating or other The specificity of MG and pyronin for staining DNA forms of pretreatment. To examine the relationship between and RNA, respectively, was argued in the earlier half of the MG staining and DNA strands, we studied the changes in 20th century. Kurnick showed that highly “polymerised” MG staining for DNA by using dot blotting on membrane DNA was stained weakly by pyronin at one-fifth intensity of in vitro. We presented here the finding that the staining RNA, and that the DNA-histone was also stained weakly as intensity of MG changes in relation to the conditions of the one-sixth of intensity. “Depolymerised” DNA was, how- DNA strands. Correspondence to: Shinobu Umemura, Department of Pathology, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259–1193, Japan. 361 362 Umemura et al. II. Materials and Methods DNA treated by DNase Two types of DNA samples were treated by DNase Methods either mildly or strongly as follows: A sample was prepared The principle and strategy of the method is based on by commercially available “activated DNA” (Worthington those of dot blot hybridization. The protocol was briefly Biochemical Corp., Freehold, NJ), by exposure of 250 g described as follows: DNA samples were applied on the DNA to 5P104 g DNase [1]) at 37LC for 15 min. This filter with the aid of a vacuum manifold with dot-like spaces DNase treatment improves the DNA as a primer for E. coli for each sample. The transfer membrane (Immobilon-N, polymerase, but is not enough to produce acid-soluble Millipore, Bedford, MA) was prepared by immersing in nucleotides. This sample was used as DNA mildly treated by 100% methanol for 20 sec and washed in distilled water DNase. The other sample was calf thymus DNA digested (DW). The membrane was attached to dot blot apparatus by DNase I (Stratagene, LaJolla, CA) under the condition (Bio-Rad Lab., Hercules, CA) with manufactured papers. for complete digestion of DNA strands, that is, an exposure DNA samples were prepared as described below, and 50– of 250 g DNA to 250 U DNase I at 37LC for 10 min. This 100 l was applied for each dot. The samples were incubated sample was applied as DNA treated by DNase strongly. The at room temperature (RT) for 5 min, vacuumed by running DNA samples treated by DNase were blotted on the mem- water, and then washed. The detached membrane was im- brane for MG staining, and examined by loading on the 2% mersed in DW, air-dried, and baked at 80LC for 2 hr to fix agarose gel with a marker of /Hind III. the DNA samples on the membrane. The membrane was stained by Veronal-Acetate buffered 1% MG (pH 4.0) for Oligonucleotide DNA strands 10 min, and then washed well in DW and air-dried. The inten- Oligonucleotides in 30 mer length were synthesized. sity of staining and changes of color tones were examined. To avoid dimer or loop formation between each DNA strand, oligonucleotides were made for poly T, poly A, poly DNA samples C and poly G separately. These oligonucleotides were Double-stranded DNA applied separately, or mixtures of poly A and poly T, and Calf thymus DNA (Worthington Biochemical Corp., poly C and poly G diluted in DW or 2P SSC buffers were Freehold, NJ) was diluted at the concentrations of 2.0, 1.0, hybridized in a tube at melting temperature, 37.0LC and 0.5, 0.25, and 0.125 mg/ml by different kinds of solutions. 80LC, respectively, and applied on to the filter. The DNA was diluted in distilled water (DW), sodium chlo- P ride-sodium citrate (SSC) buffer, pH 7.0, 10 SSC contains III. Results 3 M sodium chloride and 0.3 M sodium citrate, or 0.01 M phosphate buffered saline (PBS), and used as double-strand- Conditions of DNA strands and MG staining ed DNA. Double-stranded DNA in different buffers DNA diluted in highly concentrated SSC (10P, 5P) was Denatured single-stranded DNA and re-hybridized double- stranded DNA Calf thymus DNA diluted by DW or 2P SSC at the con- centration of 2.0 mg/ml was heated at 100LC for 5 min, and then immediately cooled in an ice water bath, and applied as denatured single-stranded DNA. The heated DNA samples were placed at room temperature (RT), and applied as re- hybridized DNA for the following time courses (30 min, 90 min and 4 hr). Scanning electron microscopic study for DNA strands DNA samples right after heating and 60 min after heat- ing were examined by scanning electron microscope. DNA samples were diluted in a solution containing 100 mg/ml cytochrome C at a concentration of 2 g/ml. DNA molecules with negatively charged phosphate base were spread to the positively charged network of mono-layered protein, cyto- chrome C, at the water surface. DNA samples diluted in DW were used for this experiment, and no salt-containing Fig. 1. Methyl green (MG) staining for double stranded DNA in solutions of PBS or SCC were applied. DNA samples different solutions. Calf thymus DNA was diluted by different solutions from the concentration of 2.0 mg/ml to 0.125 mg/ml, and were transferred onto the grid by a spreading method, and blotted on transfer membrane, and then stained by MG. The solu- observed after shadowing for enhancement. tions used for dilutions were 10P, 5P, 2P SSC, 0.01 M PBS and DW (see Materials and Methods). The solutions without DNA were also blotted and stained. Methyl Green Staining In Vitro 363 Fig. 2. MG staining for denatured single-stranded DNA and re-hybridized double-stranded DNA. DNA samples diluted in 2P SSC (a) and in DW (b) were denatured by heating, then allowed to sit at room temperature to re-hybridize. Samples were blotted on the membrane, before heat- ing, right after heating, 30 min, 1 hr 30 min and 4 hr later heating. Fig. 3. Scanning microscopic observation of DNA strands. Denatured DNA strands right after heating (A) are thinner than re-hybridized DNA at 60 min after heating (B). Bar200 nm. 364 Umemura et al. intensity, which was detected more clearly in the samples diluted by 2P SSC (Fig. 2). Attempts to observe the constructive changes of DNA strands after heating were carried out using a scanning elec- tron microscope. DNA samples right after heating showed sparse distribution of DNA strings (Fig. 3A). In contrast, re- hybridized DNA strands 60 min after heating formed tangles with increased thickness (Fig. 3B). DNA treated with DNase Staining intensities of DNA samples treated by DNase were compared. Mild treatment by DNase showed remark- ably decreased staining intensity of MG (Fig.