Rat anti mira

Immunostainings in the pupal and adult brain were carried out following standard procedures. Primary antibodies used were: rabbit-anti-GFP at 1:250 (Molecular Probes); rabbit anti-DsRed at 1:100 (Clontek); goat anti-Toll-1 at 1:10 (Santa Cruz); anti-FoxO at 1:500 (gift of P. Leopold); Guinea pig -anti-Dpn at 1:1000 (gift of Y.Jan); rat anti-Mira at 1:10 (abcam); mouse-anti-Repo 1:10 (Developmental Studies Hybridoma Bank, DSHB); rat-anti-Elav at 1:250 (DSHB). Secondary antibodies used: Alexa Donkey-anti-Rabbit 488 at 1:250; Alexa Goat-anti-Rabbit 546 at 1:250; Alexa Goat-anti Guinea-pig 633 at 1:250; Alexa Goat-anti-Rat 647 1:250; Alexa Goat-anti-mouse 647 at 1:250.

Larval and pupal brains were dissected out in phosphate buffered saline (PBS), fixed for 20 min in 4% formaldehyde in PBS and rinsed in 0.2% PBST (PBS + 0.2% TritonX‐100). For immunostaining, brains were incubated with primary antibodies overnight at 4°C, followed by an overnight secondary antibody incubation at 4°C. Samples were mounted in 80% glycerol in PBS for image acquisition. The primary antibodies used were mouse anti‐Mira (1:50; gift of Alex Gould), rat anti‐Mira (1:100, Abcam), rat anti‐pH3 (1:500; Abcam), chick anti‐GFP (1:2000; Abcam), rabbit anti‐RFP (1:100, Abcam), mouse anti‐Repo (1:50, DSHB), guineapig anti‐Hth (1:100, Claude Desplan), mouse anti‐Ey (1:50, DSHB), guineapig anti‐Slp (1:500, Kuniaki Saito), rabbit anti‐D (1:1,000, Steve Russell), guineapig anti‐Tll (1:100, Kuniaki Saito), rabbit anti‐Tll (1:100, Kuniaki Saito) and guineapig anti‐Toy (1:50, Uwe Walldorf). Secondary donkey antibodies conjugated to Alexa 555 and Alexa 647, and goat antibody conjugated to Alexa 405, 488, 555 and 647 (Molecular Probes) were used at 1:500.