Journal: Chromosome Research

Article Title: Association of modified cytosines and the methylated DNA-binding protein MeCP2 with distinctive structural domains of lampbrush chromatin

doi: 10.1007/s10577-012-9324-x

Figure Lengend Snippet: Immunostaining of Xenopus laevis LBC with 5-methylcytosine mAb. At lower magnifications a general chromomeric staining for 5mC is evident that is proportional to the DNA concentration indicated by DAPI staining. The region shown at higher magnification in the insets is indicated by the red box in the DIC image. Arrowheads in the insets indicate two of the 5mC-stained fibrils that project laterally from the chromomeric axis and that presumably correspond to the bases of some lateral loops. Note that the amplified rDNA that can be specifically detected in the fibrillar centres of extrachromosomal nucleoli by DAPI staining ( arrow ) appears unstained for 5mC, consistent with the lack of methylation in amplified rDNA determined by biochemical analyses (Dawid et al. )

Article Snippet: Primary antibodies were diluted in 10 % foetal calf serum as follows: mAb H5 (Warren et al. ) culture supernatant 1:50 dilution; α-RPC15 (Murphy et al. ) 1:500; mAb No34 (Murphy et al. ) 1:500; α-5mC mAb 33D3 (Active Motif) 1:200; α-5hmC mAb 59.1 (Active Motif) 1:4000; α-5hmC pAb (Active Motif 39769) 1:4000.

Techniques: Immunostaining, Staining, Concentration Assay, Amplification, Methylation

Journal: Chromosome Research

Article Title: Association of modified cytosines and the methylated DNA-binding protein MeCP2 with distinctive structural domains of lampbrush chromatin

doi: 10.1007/s10577-012-9324-x

Figure Lengend Snippet: Overview of 5mC-immunostaining of axolotl LBCs. a In the portion of a single LBC bivalent shown here a predominantly chromomeric staining pattern that parallels the DAPI staining intensity is apparent under low magnification. The bulk of the loop chromatin, as indicated by pol II (mAb H5) immunostaining appears not to show general 5mC immunostaining. b Regions of centromeric heterochromatin are reflected by the appearance of loopless chromatin bars ( brackets )—the examples here are close to the HLB loci ( arrowheads ) on LBC6. The intensity of 5mC in the heterochromatin appears similar to that of the DAPI staining, as in flanking chromomeres, suggesting that 5mC is not enriched in lampbrush heterochromatin. Note some laterally projecting fibrils of 5mC staining are apparent in neighbouring regions even at this low magnification (two examples indicated by arrows )

Article Snippet: Primary antibodies were diluted in 10 % foetal calf serum as follows: mAb H5 (Warren et al. ) culture supernatant 1:50 dilution; α-RPC15 (Murphy et al. ) 1:500; mAb No34 (Murphy et al. ) 1:500; α-5mC mAb 33D3 (Active Motif) 1:200; α-5hmC mAb 59.1 (Active Motif) 1:4000; α-5hmC pAb (Active Motif 39769) 1:4000.

Techniques: Immunostaining, Staining

Journal: Chromosome Research

Article Title: Association of modified cytosines and the methylated DNA-binding protein MeCP2 with distinctive structural domains of lampbrush chromatin

doi: 10.1007/s10577-012-9324-x

Figure Lengend Snippet: Detail of 5mC-immunostaining of axolotl LBC loops. a Laterally projecting 5mC-stained fibrils can be seen emerging from the brightly stained chromomeres of the chromosome axis. The fibrils often appear to be contiguous with, but not to overlap, the transcribed regions of lateral loops that are indicated by pol II staining (mAb H5). One example is indicated by the arrow. b Detail of interstitial 5mC immunostaining of lateral loops in axolotl LBCs. Short-stained regions of methylated DNA ( green in the merge) again appear contiguous with, not to overlap, the pol II-transcribed regions ( red ). Correspondingly, the loop pol II staining pattern exhibits gaps, with the gaps also matching discontinuities in the nascent RNP matrix (phase contrast image). These features are indicated by arrows for one such region in one particular loop, which interestingly appears to comprise a single matrix unit/transcription unit

Article Snippet: Primary antibodies were diluted in 10 % foetal calf serum as follows: mAb H5 (Warren et al. ) culture supernatant 1:50 dilution; α-RPC15 (Murphy et al. ) 1:500; mAb No34 (Murphy et al. ) 1:500; α-5mC mAb 33D3 (Active Motif) 1:200; α-5hmC mAb 59.1 (Active Motif) 1:4000; α-5hmC pAb (Active Motif 39769) 1:4000.

Techniques: Immunostaining, Staining, Methylation

Journal: Nature cell biology

Article Title: De novo DNA methylation drives 5hmC accumulation in mouse zygotes

doi: 10.1038/ncb3296

Figure Lengend Snippet: Tet3 is not required for loss of 5mC in early zygote. ( a ) Scheme of the targeting strategy used to generate Tet3 conditional mice. ( b ) RT-qPCR analysis of Tet3 mRNA (exon3 and exon11) in control (Tet3 mat+ ) and Tet3-depleted oocytes (Tet3 mat− ). Results are normalised to endogenous H3f3a and to control (Tet3 mat+ ). Bars represent the mean of 3 technical replicates. ( c ) Tet3 WT (n=10) and Tet3 mat−/pat+ (n=8) zygotes were stained for Tet3 protein. Quantification is represented as the mean of intensity on the paternal pronuclei after background subtraction. ( d ) Tet3 WT and Tet3 mat−/pat+ zygotes were co-stained for 5mC and 5hmC at different time points post-fertilisation. Quantification of both DNA modifications is presented as a ratio of paternal over maternal signal intensity. Each data point represents an independent zygote (n=5 PN3, n=6 PN3L and n=13 Tet3 WT zygotes; n=5 PN3, n=6 PN3L and n=19 Tet3 mat−/pat+ zygotes; 2 independent experiments). ( e ) Quantification of 5mC/dG and 5hmC/dG in Tet3 WT and Tet3 mat−/pat+ zygotes (with polar bodies) by LC/MS. Each point represents the mean of 2 technical replicates of a pool of about 100 oocytes or embryos. ( f ) Quantification of DNA modifications in 2-cell embryos derived from Tet3 WT or Tet3 mat−/pat+ zygotes analysed by LC/MS. Each point represents the mean of 2 technical replicates of a pool of about 50 embryos. Statistical analysis was carried out using Student’s t -test (two-sided). Error bars indicate s.d. PN3L, late PN3; ♀, female pronucleus; ♂, male pronucleus. n.d., non-detectable; *, p <0.05; **, p <0.01; ****, p <0.0001 (Scale bars, 5um.)

Article Snippet: XRCC1 (Serotec) 1:200, 5mC (clone 33D3, Diagenode) 1:5000 (0.02 ug/ml), 5hmC (Active Motif) 1:500 (2 ug/ml), H3K9me2 (07-441, Upstate) 1:400, H3K27me3 (gift from Dr. T. Jenuwein) 1:500, H3K4me2 (07-030, Upstate) 1:500, H3K36me3 (gift from Dr. H. Kimura,) 1:50, TET3 (C-term, Abcam) 1:200, Dnmt3a (Imgenex, IMG-268A) 1:200, Dnmt3L (Abnova, PAB2230) 1:100.

Techniques: Quantitative RT-PCR, Control, Staining, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Journal: Nature cell biology

Article Title: De novo DNA methylation drives 5hmC accumulation in mouse zygotes

doi: 10.1038/ncb3296

Figure Lengend Snippet: 5hmC and 5mC kinetics during mouse zygotic development. 5mC ( a ) and 5hmC ( b ) enrichment in mouse zygotes at different developmental stages as in assessed by immunofluorescence using 5mC and 5hmC specific antibodies. DNA is stained using PI. Representative images are shown and correspond to the 5mC and 5hmC signals quantification presented in (c). ( c ) Quantification of 5mC (red line, left axis) and 5hmC (green line, right axis) staining is shown as a ratio between signal from paternal pronucleus relative to the signal from maternal pronucleus. Values are plotted against the area of the mid-sections of the paternal pronuclei. Each data point represents a zygote. Experiment reproduced 3 times (n>100) ( d ) Loss of paternal 5mC and accumulation of 5hmC are temporally separated. Early PN3 zygotes do not show any detectable 5mC or 5hmC in paternal pronucleus. 5mC, 5-methylcytosine; 5hmC, 5-hydroxymethylcytosine; PN, pronuclei; PI, propidium iodide; ♀, female pronucleus; ♂, male pronucleus; pb, polar body. (Scale bars, 5um.)

Article Snippet: XRCC1 (Serotec) 1:200, 5mC (clone 33D3, Diagenode) 1:5000 (0.02 ug/ml), 5hmC (Active Motif) 1:500 (2 ug/ml), H3K9me2 (07-441, Upstate) 1:400, H3K27me3 (gift from Dr. T. Jenuwein) 1:500, H3K4me2 (07-030, Upstate) 1:500, H3K36me3 (gift from Dr. H. Kimura,) 1:50, TET3 (C-term, Abcam) 1:200, Dnmt3a (Imgenex, IMG-268A) 1:200, Dnmt3L (Abnova, PAB2230) 1:100.

Techniques: Immunofluorescence, Staining

Journal: Nature cell biology

Article Title: De novo DNA methylation drives 5hmC accumulation in mouse zygotes

doi: 10.1038/ncb3296

Figure Lengend Snippet: Small molecule inhibition of Tet protein activity abrogates 5hmC formation but does not prevent DNA demethylation. ( a ) 5mC and 5hmC staining of control and DMOG-treated zygotes (IVF). Quantification of both DNA modifications is represented as a ratio between the pronuclear signals (pat/mat). For 5mC staining, n=18 PN3 zygotes and n=40 PN4-5 zygotes; for 5hmC staining, n=17 PN3 zygotes and n=48 PN4-5 zygotes. This experiment has been replicated 4 times independently. ( b ) Quantification of 5mC/dG and 5hmC/dG ratio in sperm, MII oocytes, and in zygotes without polar bodies (control or treated with DMOG) by LC/MS (n=3 independent experiments with 2 technical replicates each, except for DMOG-treated zygotes; replicate of this experiment in ). Limits of quantification are summarised in . For peaks below quantification limit, an overestimation of 5hmC/dG ratio is calculated based on the limit of detection of 5hmC. ( c ) Quantification of DNA modifications in 2-cell embryos derived from DMOG-treated or control zygotes analysed by LC/MS (n=2 independent experiments and 2 technical replicates for each point). Statistical analysis was carried out using Student’s t -test (two-sided). Error bars indicate s.d. DMOG, dimethyloxallyl glycine; ♀, female pronucleus; ♂, male pronucleus. n.d., non-detectable; ***, p <0.001. (Scale bars, 5um.)

Article Snippet: XRCC1 (Serotec) 1:200, 5mC (clone 33D3, Diagenode) 1:5000 (0.02 ug/ml), 5hmC (Active Motif) 1:500 (2 ug/ml), H3K9me2 (07-441, Upstate) 1:400, H3K27me3 (gift from Dr. T. Jenuwein) 1:500, H3K4me2 (07-030, Upstate) 1:500, H3K36me3 (gift from Dr. H. Kimura,) 1:50, TET3 (C-term, Abcam) 1:200, Dnmt3a (Imgenex, IMG-268A) 1:200, Dnmt3L (Abnova, PAB2230) 1:100.

Techniques: Inhibition, Activity Assay, Staining, Control, Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Journal: Nature cell biology

Article Title: De novo DNA methylation drives 5hmC accumulation in mouse zygotes

doi: 10.1038/ncb3296

Figure Lengend Snippet: Hydroxylation targets newly deposited 5mC generated by Dnmt3a and Dnmt1. ( a ) Isotope-labelled 5mC (5mC*) quantified by LC/MS after incubation of zygotes with heavy methionine ( 13 C,d 3 -methyl) in the presence or absence of aphidicolin (IVF). Each point represents a biological replicate (n=2). An example of the 5mC* peak detected by LC/MS is depicted for each condition and further confirms the existence of both maintenance and de novo DNA methylation in zygotes. Note that the observed signal represents only a fraction of new zygotic 5mC due to the endogenous pool of unlabelled S-adenosyl-methionine. ( b ) Inhibition of new zygotic DNA methylation by 5-azadeoxycytidine (azadC) (IVF) affects accumulation of paternal 5hmC as assessed by staining using 5mC and 5hmC specific antibodies. Only zygotes with a paternal mid-section area > 200um 2 (~PN4-5 zygotes) were considered to avoid developmental staging bias. Quantification of 5mC and 5hmC is represented as signal intensity in paternal and maternal pronuclei (left axis) or as a ratio between the pronuclei signal (pat/mat) (right axis). (n=6 control and n=18 treated zygotes; experiment replicated twice independently). 5mC and 5hmC staining in PN4-5 zygotes (paternal mid-section area >200um 2 ) with maternal ( c ) Dnmt3a ([♀ Dnmt3a 2lox/2lox , Zp3-Cre ] × ♂WT) (n=12 WT and n=10 KO zygotes; experiment reproduced twice independently) or ( d ) Dnmt1 ([♀ Dnmt1 2lox/2lox , Zp3-Cre × ♂WT]) (n=13 WT and n=11 KO zygotes; experiments reproduced twice independently) deletion. Note that only total signal intensity is plotted in (c) as Dnmt3a deletion affects 5mC and 5hmC level in maternal PN. ( e , f ) Tet3 localisation and signal intensity is identical between ( e ) WT (n=4) and Dnmt3a KO (n=3) or ( f ) WT (n=5) and Dnmt1 KO (n=3) zygotes. Quantification is represented as the mean of intensity on the paternal pronuclei after background subtraction. Statistical analysis was carried out using Student’s t -test (two-sided). Error bars indicate s.d. *, p <0.05; **, p <0.01; ***, p <0.001. ♀, female pronucleus; ♂, male pronucleus; azadC, 5-azadeoxycytidine. (Scale bars, 5um.)

Article Snippet: XRCC1 (Serotec) 1:200, 5mC (clone 33D3, Diagenode) 1:5000 (0.02 ug/ml), 5hmC (Active Motif) 1:500 (2 ug/ml), H3K9me2 (07-441, Upstate) 1:400, H3K27me3 (gift from Dr. T. Jenuwein) 1:500, H3K4me2 (07-030, Upstate) 1:500, H3K36me3 (gift from Dr. H. Kimura,) 1:50, TET3 (C-term, Abcam) 1:200, Dnmt3a (Imgenex, IMG-268A) 1:200, Dnmt3L (Abnova, PAB2230) 1:100.

Techniques: Generated, Liquid Chromatography with Mass Spectroscopy, Incubation, DNA Methylation Assay, Inhibition, Staining, Control

Journal: Epigenetics

Article Title: De novo methylation in male germ cells of the common marmoset monkey occurs during postnatal development and is maintained in vitro

doi: 10.1080/15592294.2016.1248007

Figure Lengend Snippet: Global DNA methylation is gradually established in germ cells during postnatal development. (A-D) Representative micrographs show 5mC stainings of testicular cross sections from neonatal, 4-months-old, 8-months-old, and adult animals. Arrowheads indicate gonocytes, arrows spermatogonia, empty arrows spermatocytes and empty arrow heads round spermatids. Positive cells are indicated by black and negative cells by blue symbols. No staining was detected in corresponding IgG controls (Fig. S1). Scale bars represent 50 µm. (E) Proportion of 5mC positive germ cells in histological stainings (n = 3 per age group). Data are presented as mean with standard deviation. Individual values for each biological replicate are indicated by dots. Statistically significant differences between all 4 groups were found applying the non-parametric Kruskal Wallis test (P < 0.05).

Article Snippet: Detection of 5mC positive cells was performed with the primary mouse-anti-5mC antibody (C15200081, Clone: 33D3, Lot GF-004, Diagenode, dilution: 1:100), the secondary chicken-anti-mouse IgG-Biotin antibody (sc-2985, Santa Cruz Biotechnology, dilution 1:100), and streptavidin conjugated with HRP (S5512, Sigma-Aldrich, dilution: 1:500).

Techniques: DNA Methylation Assay, Staining, Standard Deviation

Journal: Epigenetics

Article Title: De novo methylation in male germ cells of the common marmoset monkey occurs during postnatal development and is maintained in vitro

doi: 10.1080/15592294.2016.1248007

Figure Lengend Snippet: Global DNA methylation pattern during postnatal development in specific germ cell types. The proportion of 5mC positive cells was determined separately for each cell type in neonatal, 4-months-old, 8-months-old, and adult marmosets (n = 3 per age group). Significant differences between values for spermatogonia at the different developmental stages and for the different cell types in the adult testis were found applying the non-parametric Kruskal Wallis test (P < 0.05). Gon: gonocytes, Spg: spermatogonia, Spc: spermatocytes, Rs: round spermatids, Es: elongated spermatids.

Article Snippet: Detection of 5mC positive cells was performed with the primary mouse-anti-5mC antibody (C15200081, Clone: 33D3, Lot GF-004, Diagenode, dilution: 1:100), the secondary chicken-anti-mouse IgG-Biotin antibody (sc-2985, Santa Cruz Biotechnology, dilution 1:100), and streptavidin conjugated with HRP (S5512, Sigma-Aldrich, dilution: 1:500).

Techniques: DNA Methylation Assay

Journal: PLoS ONE

Article Title: Global DNA methylation of peripheral blood leukocytes from dogs bearing multicentric non-Hodgkin lymphomas and healthy dogs: A comparative study

doi: 10.1371/journal.pone.0211898

Figure Lengend Snippet: 1: quantity of dC. 2: quantity of 5MeCyt. The curve area yields the numerical values to be entered into the formula for calculating the percentage of overall DNA methylation (5MeCyt%).

Article Snippet: CBs produced by leukocytes from control and neoplastic dogs were submitted to the same protocol previously described for immunophenotyping, but the anti-5-methylcytosine (5MeCyt) (33D3, 1:100) (Abcam, Cambridge, MA, USA) was used as primary antibody.

Techniques: DNA Methylation Assay

Journal: PLoS ONE

Article Title: Global DNA methylation of peripheral blood leukocytes from dogs bearing multicentric non-Hodgkin lymphomas and healthy dogs: A comparative study

doi: 10.1371/journal.pone.0211898

Figure Lengend Snippet: Control and experimental groups for global DNA methylation quantification by HPLC and immunoreactivity for 5MeCyt.

Article Snippet: CBs produced by leukocytes from control and neoplastic dogs were submitted to the same protocol previously described for immunophenotyping, but the anti-5-methylcytosine (5MeCyt) (33D3, 1:100) (Abcam, Cambridge, MA, USA) was used as primary antibody.

Techniques: DNA Methylation Assay, High Performance Liquid Chromatography, Standard Deviation

Journal: PLoS ONE

Article Title: Global DNA methylation of peripheral blood leukocytes from dogs bearing multicentric non-Hodgkin lymphomas and healthy dogs: A comparative study

doi: 10.1371/journal.pone.0211898

Figure Lengend Snippet: Different grades of staining intensity caused by anti-5-methylcytosine antibody. A: strong immunoreactivity and B: weak immunoreactivity (40x objective).

Article Snippet: CBs produced by leukocytes from control and neoplastic dogs were submitted to the same protocol previously described for immunophenotyping, but the anti-5-methylcytosine (5MeCyt) (33D3, 1:100) (Abcam, Cambridge, MA, USA) was used as primary antibody.

Techniques: Staining