Software las x 3

Briefly, male larval heads (heads refers to anterior 1/3 of larvae) were dissected off in 1X PBS and inverted so that salivary glands (still attached) were exposed, and placed into an Eppendorf tube on ice containing 1X PBS. Tissue was fixed in 4% PFA in PEM buffer (100mM PIPES pH 7, 1mM EGTA pH 8, 2mM MgSO₄) for 20 mins on a nutator at RT. After fixation, heads were washed 6x in 1mL BBT (1% (w/v) BSA, 0.1% (v/v) Triton X-100 in PBS). Heads were incubated overnight on a rotator in primary antibody diluted in BBT (Mtor, 1:30) at 4C. On the second day, heads were washed for 30 mins in BBT, changing buffer 6x. After washes, the appropriate secondary antibody, diluted in BBT was added and incubated overnight on a rotator at 4C. On day 3, heads were washed for 30 mins in 0.1% PBT, changing buffer 6x. DNA staining with Hoechst was carried out at 1:1000 in PBT on a covered nutator for 5 mins, then removed and washed with PBT. Samples were washed 3 more times with PBT. To mount samples, heads were transferred to a glass dissecting dish, salivary glands were separated off and placed onto a slide in a drop of vectashield, and a coverslip was placed on top. Slides were allowed to dry for ~30 mins before sealing with nail polish. Confocal imaging was conducted at room temperature on a Leica TCS SP8 Confocal using PL APO 20X, and Leica Software LAS-X 3.3.