Ovary Dissection and Fixation Protocol

Ovaries from five females were dissected in Robb’s medium (100 mM HEPES pH 7.4; 40 mM potassium acetate; 10 mM glucose; 1 mM CaCl₂; 55 mM sodium acetate; 100 mM sucrose; 1.2 mM MgCl₂) and ovarioles fixed for 10 min in 100 µl fixation solution (200 mM Cacodylate pH 7.2; 80 mM potassium acetate pH 7.5; 20 mM EGTA; 200 mM sucrose; 20 mM sodium acetate pH 5.2) at room temperature. DNA was counterstained with 100 µl DAPI solution (0.4 µg/ml; Sigma) overnight. The following day, ovarioles were transferred into mounting medium (2.5% propyl gallate w/v; 85.5% glycerol) and mounted onto slides.