Antirabbit igg conjugated with alexa fluor 594

Fresh-frozen tissue sections were cut onto Superfrost/Plus slides (Thermo Fischer Scientific, Waltham, MA, USA). A negative control was conducted for each run. For immunofluorescence, cells were grown in eight-well TC Lab-Tek Chamber Slides (Thermo Fischer Scientific). The slides were incubated with the primary antibody overnight at 4 C. Intracellular markers were stained with the primary antibody, followed by antimouse IgG conjugated with Alexa Fluor 488 and/or antirabbit IgG conjugated with Alexa Fluor 594 (Invitrogen). The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were recorded using a Leica DFC310 FX microscope.

Dissection of 3–7 day old adult flies and immunohistochemistry of the adult nervous system were carried out as described previously^{48 (link)} with some modifications in primary and secondary antibodies. Dissection of male reproductive organs was performed on Sylgard plate covered with 1 × PBS. To obtain male genitalia, the lower half of the abdomen were dissected and fixed in 4% PFA at room temperature for 20–30 min. After which the PFA was replaced with 1xPBS. The genitalia were cut out with a pair of microscissors to ensure a flat surface for mounting. Dissected samples were fixed and proceeded with immunostaining as described previously^{48 (link)}. The following primary and secondary antibodies were used in this study: rat polyclonal anti-mCD8 (1:100; Caltag, Burlingame, CA), mouse anti-GFP (1:500, Life technologies), mouse anti-nc82 (1:20; Hybridoma Bank)^{49 (link)}, rabbit anti-Dsx^M (1:2000) (kindly supplied by Brian Oliver laboratory, NIH), anti-rat IgG conjugated with Alexa Fluor 488 (1:300; Invitrogen), anti-mouse IgG conjugated with Alexa Fluor 488 (1:300 Invitrogen), and anti-rabbit IgG conjugated with Alexa Fluor 594 (1:300; Invitrogen).