Figure 1.
The influence of primary antibody incubation time and temperature on the level of staining of the 5hmC and 5meC antigens in the male and female pronuclei of mouse PN5 stage zygotes.
Zygotes were collected directly from the oviduct, fixed and prepared for staining after either acid pretreatment (5hmC or acid plus trypsin pretreatment (5meC). Zygotes were incubated with primary antibodies for between 1 and 18 h at either room temperature or 4°C. Staining levels in the male and female pronuclei of each zygote was assessed as the optical density of staining in each pronuclei. The data is shown as the mean intensity/pixel within each pronuclei (top panel) and the total staining of all pixels within each pronuclei (bottom panel). The data is expressed as the SEM of arbitrary units of optical density measured (AU). The number of zygotes measure for each treatment (n = ) is shown above each bar. The zygotes were collected from three independent replicates. Differences in staining levels at individual times were assessed by paired T-tests, *P<0.05; **P<0.01; and ***P<0.001. The changes in staining levels relative to time were assessed by univariate analysis using the general linear model and significant effects are noted within the results text. In each case conditions for staining and imaging were managed so that no signal was detected from the non-immune negative control treatments, and these same conditions were used for imaging primary antibody staining.
Figure 2.
The effect of antigen retrieval procedures on the detection of 5hmC and 5meC in the mouse PN5 stage zygote.
Zygotes were collected directly from the oviduct and fixed and prepared for staining. After antigen retrieval steps, zygotes were incubated with primary antibodies for 1 h at room temperature (5hmC) or 18 h at 4°C (5meC) and each primary antibody was detected by an FITC-labeled secondary antibody. The pronuclei were counter-stained for DNA with PI and the FITC and DNA images merged. Three epitope retrieval approaches were under taken: −H-T, zygotes were not pretreated with either acid (HCI) or trypsin; +H-T, they were pretreated with acid only; and +H+T, epitope retrieval was by a combination of acid and trypsin treatment. The images are representative of three independent replicates. In each case conditions for staining and imaging were managed so that no signal was detected from the non-immune negative control treatments, and these same conditions used for imaging primary antibody staining. The bar is 5 µm and all images are at the same magnification.
Figure 3.
Staining of 5hmC with a monoclonal antibody.
PN5 stage zygotes were collected directly from the oviduct and fixed and prepared for staining. They incubated with anti-5hmC or non-immune IgG for 1 h at room temperature (RT) or 18 h at 4°C after epitope retrieval by acid. Three representative images of each treatment are shown. These are representative of three independent replicates with more than 30 embryos per treatment in each replicate. The bar is 5 µm and all images are at the same magnification.
Figure 4.
The effects of incubation time, temperature and epitope retrieval strategies on the simultaneous staining of 5meC and 5hmC.
PN5 stage zygotes were collected directly from the oviduct and fixed and prepared for staining. They were co-incubated with anti-5meC and anti-5hmC for 1 h at RT or 18 h at 4°C after epitope retrieval by either acid and trypsin treatment (+H+T) or acid treatment alone (+H-T). 5meC was detected with an FITC-labeled secondary antibody (green) and 5hmC by a Alexa Fluor633 (red) label. The images are representative of at least three independent replicates. The signals from the green and red channels were merged to detect co-localization. The bar is 5 µm and all images are at the same magnification.
Figure 5.
The patterns of 5meC and 5hmC staining in each pronuclei during zygotic maturation.
Zygotes were collected directly from the oviduct at various times after fertilization and analyzed at the PN1-5 stages of development. Zygotes were subjected to antigen retrieval by both acid and trypsin treatment and were co-stained with anti-5meC and anti-5hmC for 18 h at 4°C. Anti-5meC was detected in the green channel and anti-5hmC in the red channel. The outputs from these channels were merged to show the areas of co-localization. For each primary antibody determined that provided no signal for the non-immune control immunoglobulin and these conditions used for detection of primary antibody. The same conditions for imaging were used for each stage of maturation. The results are representative of 3 independent replicates.
Figure 6.
Analysis of heterogeneity in co-staining of 5meC and 5hmC in the male and female pronuclei of PN5 stage zygotes.
Zygotes were collected directly from the oviduct and fixed and stained as in Fig. 4. Matched male and female pronuclei from individual zygotes are shown. Each image is a 1.03 µm equatorial cross-sectional image of each pronuclei. The image analysis program was then used to measure the optical density of staining of each antigen along the plane shown by the white line. Each corresponding graph shows the optical density per pixel (Y-axis, arbitrary units) along this plane axis, number of pixels). The green line on graphs shows intensity of 5meC staining and the red line is 5hmC staining along this path. Examples of areas where localized increases in 5meC staining was not accompanied by a similar magnitude increase in 5hmC staining are marked by (X) and accumulation of 5hmC relative to 5meC is marked by (*). Examples of nucleolar precursor bodies are shown by white arrows. The corresponding male and female pronuclei from each zygote were imaged and analyzed under identical conditions.