Dissection and Immunostaining and HCR-FISH

All animals were dissected as adults at 24 hours past mid L4 unless otherwise mentioned. Germlines were dissected and immunostained as described previously⁶⁵ with the following alterations. Fixation was performed in 3% PFA for 15 min. Methanol treatment was performed overnight. For HCR-FISH, dissections were performed as above. After methanol treatment, germlines were processed as previously described³⁶ with the following alterations. Processing was performed in the same tubes as immunostaining (6mm tube) and all wash incubation volumes were halved and DAPI was added during the final washes. Additionally, amplification hairpins were added at ½ of the recommended concentration. In the case that there were two treatments for a particular experiment, probe hybridization was performed on one of the treatments using probes against either mpk-1 or cyb-1 to differentiate between treatments. That sample was then washed with probe wash buffer as in the protocol, and then two conditions were combined and probe hybridization was performed for the experimental probes.