Anti-OOEP antibody validated using mouse oocytes isolated from 8 week old FVB/N mice. Oocytes were fixed in 2% PFA (+ 100 mM Hepes, 50 mM EGTA (pH7), 10 mM MgSO4, and 0.2% Triton-X-100) for 30 min at 37ºC. They were then extracted overnight at 4ºC in 0.2% Triton-X-100 in PBS prior to blocking and antibody incubations. Anti-OOEP antibody was used at a 1:1000 dilution (stock 1.33 mg/mL) in 5% BSA with 0.1% Triton-X-100 and samples were incubated at 4ºC overnight . Secondary antibody was goat-anti rabbit conjugated to AlexaFluor488 1:400 (stock 2mg/mL), incubated 1.5 hours at RT.
Images provided courtesy of Ida Jentoft, Max Planck Institute for Biophysical Chemistry.