Vectashield dapi

Extracted macrophages were collected by centrifugation at 500 g for 5 min at room temperature. The cell pellet was resuspended in a small volume of Phospho-buffered saline (PBS) and smeared on a cover slip. The cell suspension was left to dry before cells were fixed with 4% paraformaldehyde in 0.1M Phosphate Buffer for 20 min at room temperature. Cells were washed 3 times in 0.1M PBS and permeabilized in 0.5% Triton-X 100 in PBS. Cells were blocked for 1 hr at room temperature in 20% Fetal Bovine Serum +0.25% Triton X-100 in PBS. Primary antibodies were diluted in blocking buffer: anti-HA (Roche, Basel, Switzerland) 1:50, anti-Golgin 84, 1:25, anti-Calnexin 99a 1:25, anti-Hrs.8.2 1:25 or anti-Rab7 1:25 all from DSHB (Riedel et al., 2016 (link)), and incubated for 1 hr at room temperature. Cells were then washed 5 times in blocking buffer. Secondary antibodies were diluted in blocking buffer: anti-rat 633 1:300, anti-mouse 488 1:300 (both from ThermoFisher Scientific, Waltham, Massachusetts, USA). Secondary antibodies were incubated for 1 hr at room temperature. Cells were washed 5 times in PBS + 0.1% Triton X-100 and mounted in VectaShield + DAPI (LifeTechnologies, Carlsbad, USA) utilized at 1:75.

3–5 day old females were fed with yeast for 2 days at 25°C. For ovary dissection, females were anesthetized using the FlyNap anesthetic kit (Carolina, Burlington, NC, USA) and further transferred to ice cold PBS in which ovaries were extracted with pre-cleaned forceps. Individual ovaries were fixed in 4% Paraformaldehyde/PBS at room temperature (RT) for 20 min with agitation. Three wash steps with PBS at RT for 10 min were performed and individual ovaries were incubated in PBS supplemented with 0.1% of Triton X-100 (PBT) for 10 min at RT to allow permeabilization of the tissue. Ovaries were incubated in phalloidin-A488 (Thermo Fisher) diluted in PBT (1:300) overnight at 4°C. After being washed with PBT and PBS, ovaries were mounted in Vectashield + DAPI (LifeTechnologies, Carlsbad, USA).