Anti 5 methyl cytosine

Immunostaining was carried out as was previously described [36 (link)]. Briefly, the following rabbit monoclonal and polyclonal antibodies against modified histones and DNA were used: anti-dimethyl histone H3 at lysine 4 (1:100; Abcam, cat. no. (ab7766), anti-dimethyl histone H3 at lysine 9 (1:100; Millipore, cat. no. 07–441) and anti-5-methyl-cytosine (1:300, Abcam, cat. no. ab73938). Two secondary antibodies were also applied–Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Molecular Probes, cat. no. A-11008) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, Molecular Probes, cat. no. A-11001). The fluorescence of DAPI (excitation 405 nm, emission 425–475 nm) and Alexa488 (excitation 488 nm, emission 500–600 nm) was registered using an Olympus FV1000 confocal system (Olympus) equipped with an IX81 inverted microscope, a 60x Plan Apo oil-immersion objective lens (NA 1.35), a 50 mW, 405 nm diode laser and a 100 mW multi-line argon ion laser (Melles Griot BV). An axial series of 2-D fluorescence images of optical sections through the chromosomes (z-stacks) was collected using two separate photomultipliers (R6357, Hamamatsu). Image processing operations were performed with ImageJ (Fiji).

The immunostaining was done for 3 mM of MH-treated plants and for 225 Gy of gamma ray-treated plants. The immunostaining was carried out as previously described (Braszewska-Zalewska et al. 2010 (link), 2012 (link), 2013 (link)). Briefly, the following rabbit monoclonal and polyclonal antibodies against modified histones and DNA were used: anti-acetyl histone H4 at lysine 5 (1:100; Millipore, cat. no. 04-118), anti-dimethyl histone H3 at lysine 9 (1:100; Upstate, cat. nos. 05-768 and 07-212), anti-5-methyl-cytosine (1:300, Abcam, cat. no. ab73938). Two secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Molecular Probes, cat. no. A-11008) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, Molecular Probes, cat. no. A-11001), were applied.

Nuclei suspension for 5mC analysis was prepared as described earlier [67 (link)] with minor modifications. Tissue sections from leaves, immunostaining and image acquisition and processing were carried out as previously described [68 (link)–70 (link)]. Briefly, the following primary mouse monoclonal antibody against methylated DNA: anti-5-methyl-cytosine (1:00, Abcam, Cat. no. ab73938) and secondary antibody: Alexa Fluor 488 goat anti-mouse (Invitrogen, Molecular Probes, Cat. no. A-11001) were used. Images from leaf sections were registered using confocal laser scanning microscopy (CLSM) (Olympus FV1000) and high content screening fluorescence microscopy (HCSFM) (Scan'R, Olympus). Image processing operations were performed with ImageJ (Fiji) or the automated segmentation-based Scan'R Analysis software (Olympus). An average of 100 – 400 nuclei (depending on the method used) were analysed for each experimental group. Alexa 488 fluorescence (5mC) was segmented with the threshold value parameter, and then the fluorescence intensity was measured. The mean fluorescence intensity values were estimated in the relative units and shown in Figs. 3A and B. A paired Student's t-test was applied to check the statistically significant difference between samples.