Ovary dissection and immunostaining on cryosections

Adult zebrafish were euthanized using an overdose of tricaine, according to the institutional guidelines approved by the German authorities. The ovary was dissected out of freshly euthanized females using a sharp pair of forceps. The ovarian tissue was quickly rinsed in L-15 medium (pH 9) (Gibco, 31415-029) and then used for further processing.

For cryosections, dissected ovaries were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) overnight at 4°C and washed with PBS 5× for 5 min each. Whole ovaries were then transferred to the following solutions and allowed to equilibrate for one day: 25% sucrose in PBS, 35% sucrose in PBS, 1:1 optimal cutting temperature compound (OCT) (TissueTek): 35% sucrose in PBS and 100% OCT. The ovaries were then transferred to a mold containing fresh OCT and frozen on a metal block that was stored at −80°C. A cryotome was used to obtain 10 µm cryosections, with the block at −15°C and the chamber at −20°C, which were placed on Superfrost UltraPlus slides (Thermo Scientific, J3800AMNZ) and air-dried at room temperature (RT) for at least 2 h and then stored at −80°C.