in DMEM supplemented with 10% FBS in a CO2 incubator. The cells were briefly washed with 1xPBS and fixed in 4% PFA in PBS for 15 min at room temperature. (I) The cells were permeabilised for 10 min using PBS, 0.2% Triton X-100 and were blocked for 1 hr in PBST (PBS, 0.05% Tween20), 1% BSA at room temperature. (II) The antibody staining was carried out for 1 hr in PBST. (III) The slides were subsequently washed 36 in PBST (10 min each wash) and (IV) incubated with Alexa dye-conjugated secondary antibodies (Molecular Probes) for 1 hr at room temperature in the dark, and (VI) washed 565 min in PBST. The slides were subsequently washed for 3610 min in PBST. (VII) Cells were blocked in PBST, 1% BSA at 4uC overnight, followed by the steps II to VI for staining with germ cell specific antibodies (Oct-3/4 or Ddx4). The slides were then mounted in ProLong Gold Antifade Reagent with DAPI (Invitrogen) and imaged using IX81 inverted microscope (Olympus). During and after applying Alexa dyes conjugated secondary antibodies, specimens were kept in the dark. 5mC staining After performing the above steps I to VI for germ cell-specific antibodies (Oct-3/4 or Ddx4), the cells were permeabilized for 30 min using PBS, 0.2% Triton X-100 and were treated with RNase A (100 mg/ml) for 30 min at room temperature and washed 265 min in PBS. DNA was denatured 10 min 4 N HCl, 0.1% Triton X-100 and washed 265 min in PBS. The cells were neutralized for 30 min in 100 mM Tris HCl, pH 8.0 and washed 265 min in PBS. The cells were blocked in PBST, 1% BSA at 4uC overnight. The steps from II to VI were performed for 5mC antibody, and blocked for 1 hr in PBST, 1% BSA at room temperature. The anti-DNA antibody staining was carried out in PBST at 4uC overnight, followed by the steps from II to VI. The slides were then mounted in ProLong Gold Antifade Reagent (Invitrogen). Quantitation The quantification of staining intensity was performed using Image-Pro Plus version 6.3 software (Media Cybernetics, Carls- bad, CA). Mean fluorescence intensity/area values were measured for 20–30 germ cells (green) and 200–300 somatic cells. After background correction, red fluorescence values were divided by References 1. Reik W, Dean W, Walter J (2001) Epigenetic reprogramming in mammalian development. Science 293: 1089–1093. 2. Sasaki H, Matsui Y (2008) Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nat Rev Genet 9: 129–140. 3. Reik W (2007) Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447: 425–432. 4. Hajkova P, Erhardt S, Lane N, Haaf T, El-Maarri O, et al. (2002) Epigenetic reprogramming in mouse primordial germ cells. Mech Dev 117: 15–23. 5. 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Image quantification results obtained in two indepen- dent experiments by two investigators (A) MA and (B) ST are shown for two antibodies, H3K79me2 and H3K79me3, at 17.5 dpc. The experiments included fetal gonad collection, immunostaining, microscopy and quantification. Mean fluores- cence intensity/area values were measured for 20–30 male or female germ cells (MGC and FGC) and 200–300 male or female somatic cells (MSC, FSC). After background correction, red fluorescence values were divided by DAPI intensity values for germ cells and somatic cells separately. Standard deviation values were calculated from the individual GC values over the average SC values. Male germ cells exhibited statistically significant difference from somatic cells in global H3K79me2 and H3K79me3 staining according to T-tests (***p-value,0.000001). 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Williamson CM, Turner MD, Ball ST, Nottingham WT, Glenister P, et al. (2006) Identification of an imprinting control region affecting the expression of all transcripts in the Gnas cluster. Nat Genet 38: 350–355. PLoS ONE | www.plosone.org 11 August 2011 | Volume 6 | Issue 8 | e23848