A6455

Mammalian (pCaggs) expression vectors encoding p27^kip1, p27^kip1(ck-) and RhoA(N19) have been described previously [16 (link)], while an Rp58 expression construct with a pCIG vector which lacks a GFP cassette was used [11 ]. RNAi for Rp58 was achieved using a pool of targeting siRNAs (Dharmacon GE Life Sciences) which was previously verified for specificity of knockdown as well as a pSilCaggs-Rnd2shRNA1 vector to induce Rnd2 RNAi [11 ]. Primary antibodies used for immunostaining analysis include chicken antibody to GFP (Abcam, ab13970, 1:700), mouse anti-p27^kip1 (BD Biosciences, 1:400), rabbit anti-Rp58 (Proteintech Group, 1:250), rabbit anti-Ki67 (NCL-Ki67p, Leica, 1:1000), pHH3(ser10) (06–570, Merck Millipore, 1:1000), mouse anti βIII-tubulin (Covance, MMS-435P, 1:1000), mouse anti-Nestin (Millipore, MAB353, 1:300), rabbit anti-Pax6 (Covance, PRB-2788, 1:500), rabbit anti-Tbr2 antibody (Abcam, ab233345, 1:500), rabbit polyclonal antibody to GFP (Invitrogen, A6455, 1:1000). Alexa fluor secondary antibodies include goat anti- chicken IgG (Invitrogen, A11039, 1:700), goat anti-mouse (Invitrogen, A11031, 1:800), and goat anti-rabbit IgG (Invitrogen, A6455, 1:1000). The nuclei of cells were visualised with DAPI.

Immunofluorescence studies were performed as follows⁶⁰ : 5–6 day old larvae were dissected in phosphate buffer saline (PBS) or HL3.1 (pH 7.2)^{61 (link)}, fixated in 4% paraformaldehyde in PBS for 40 min, washed in PBS with 0.3% Triton-X 100 (PBT), and afterwards blocked in 5% normal goat serum in PBT. Specimens were incubated in primary antibody solution containing polyclonal rabbit anti-GFP antibody (A6455, Molecular Probes, dilution 1:1000) or monoclonal mouse anti-GFP (A11120, Molecular Probes, dilution 1:250), polyclonal rabbit anti-sNPFp (ref. ^{40 (link)}, dilution 1:1000) and 3% normal goat serum in PBT for one to two nights at 4 °C. Then brains were washed six times in PBT and incubated for one night at 4 °C in secondary antibody solution containing goat anti-rabbit Alexa 488 (Molecular Probes, dilution 1:250) or combined goat anti-mouse DyLight 488 (Jackson ImmunoResearch, dilution 1:250) and goat anti-rabbit Alexa 635 (Molecular Probes, dilution 1:250). Finally, specimens were rinsed six times in PBT and mounted in 80% glycerol or Vectashield mounting medium (Vector Laboratories, USA). Until scanning with a Leica SP8 confocal light scanning microscope, brains were stored in darkness at 4 °C. Image processing was performed with Fiji^{62 (link)} and Adobe Photoshop CS6 (Adobe Systems, USA).

To analyze the expression pattern of OK107-Gal4 rabbit anti-GFP antibody (A6455, Molecular Probes; 1:1000) and two different mouse antibodies for staining the cholinergic neuropil (ChAT4B1; DSHB, Iowa City, IA, 1:150) and axonal tracts (1d4 anti-Fasciclin 2; DSHB, Iowa City, IA; 1:50) were applied [71 (link), 117 ]. A specific antibody for the Synapsin protein was used to verify the mutation syn⁹⁷ (monoclonal mouse anti-syn, 3C11; DSHB, Iowa City, IA, 1:10) [54 (link)]. To analyze if the expression level of Bruchpilot is specifically reduced in the MB KCs by driving UAS-RNAi^B3C8 via OK107-Gal4, monoclonal mouse anti-nc82 was used (nc82, DSHB, Iowa City, IA, 1:10) [58 (link)]. As secondary antibodies goat anti-rabbit IgG Alexa Fluor 488 (A11008, Molecular Probes, 1:200) and goat anti-mouse IgG Alexa Fluor 647 (A21235, Molecular Probes, 1:200) were used.