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After three washes in phosphate buffered saline with 0.1% Tween (PBST hereafter) to remove residual methanol, the gonads were incubated with 50 g/ml protease K in PBST for 30 mins, followed by 15 mins re-fixation in 3% paraformaldehyde / 0.25% glutaraldehyde / 0.1 M K2HPO4 (pH7.2), three washes in PBST, 15 mins incubation in PBST with 2 mg/ml glycine to remove residue aldehyde, then another three washes in PBST prior to hybridization