Mouse anti 5mc antibody

Immunostaining of 5-mC was performed as described previously (19) . Semen samples were washed three times with PBS and smears were prepared on glass slides. The smears were fixed with ethanol (70%) at 22 C for 20 minutes and then air dried. The slides were washed twice in PBS with 0.5% Tween (PBS-T; Sigma-Aldrich) for 5 minutes at RT. To achieve decondensation of sperm DNA, slides were incubated at RT in 1 M/L HCl-Tris buffer at pH 9.5, containing 25 mM/L dithiothreitol (DTT; Sigma-Aldrich) for 20 minutes. The slides were then washed twice in PBS-T and incubated with mouse anti-5-mC antibody (Eurogentec) diluted 1:100 in PBS-T for 1 hour at RT or overnight at 4 C. The slides were washed twice in PBS-T for 5 minutes and further incubated with anti-mouse antibodies coupled with fluorescein isothiocyanate. The slides were then washed twice in PBS-T and stored at 4 C in a dark chamber until use. Control slides were prepared using cells incubated with buffer instead of primary antibody. Methylation in the samples was assessed by visualization of immunofluorescence in the head of the sperm using an epifluorescence microscope (Carl Zeiss Axiophot microscope; exciter filter BP450-490, emission filter BP520) (Fig. 1B). At least 300 sperm were analyzed on each slide and the percentages of sperm exhibiting strong, weak, and no staining were recorded.