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.