M sssi

Females guts were dissected in Schneider medium, washed in PBS, and fixed in 6% EM-grade formaldehyde (Polysciences, Warrington, PA) diluted in PBS, with three volumes of heptane for 20 min. The dissected tissue was then washed with PBS, permeabilized using 0.5% Triton X-100 in PBS for 1 hr, washed with PBS three more times and blocked in PAT (1% BSA, 0.1% Triton X-100 in PBS (PBX) overnight. The next day, guts were washed twice with 250 µl M.SssI reaction buffer supplemented with 16 µM S-Adenosyl-L-methionine, Rre-suspended in 50 µl of M.SssI reaction buffer supplemented with 25U of M.SssI (NEB, Ipswich,MA) and 16 µM S-Adenosyl-L-methionine and incubated for 1 hr at 25°C on an orbital shaker. DNA was denatured by adding 1 ml of 2N HCl for 30 min at room temperature. The solution was then neutralized in 100 mM Borax for 5 min and washed twice with PBS. Detection of 5mC was performed using anti methyl-cytosine antibody primary antibody (monoclonal anti-5-mC, clone 33D3, Active Motif, Carlsbad, CA) that was added at 1 μg/ml. The next day, guts were washed three times with PBT (0.1% BSA, 0.1% Triton X-100 in PBS). We used secondary anti-mouse used Alexa-568 (1:1000) and DNA was visualized using DAPI for 1 hr and following two washes was mounted in Fluoromount-G for confocal imaging.