Larval brains were dissected with forceps, fixed in 4% paraformaldehyde, processed, stained, and imaged as previously described (11 (link)). The following antibodies were used: 8D12 mouse anti-Repo (1:10, Developmental Studies Hybridoma Bank), anti-phospho-S21-Sqh (1:500, gift of Robert Ward) (26 (link)), anti-Anillin (1:500, gift of Maria Giansanti) (27 (link)). Secondary antibodies were conjugated to Cy3 (1:150), Alexa-488, or Alexa-647 (1:100) (Jackson Laboratories). Brains were mounted on glass slides ventral side down in vectashield and whole mount imaged on a Zeiss LSM 700 confocal system. For experiments where protein levels were compared between genotypes, all samples were prepared, subjected to immunohistochemistry, imaged, and image processed in a parallel manner side by side. 6 or more brains were stained with each Ab combination, and representative images are shown for each result. All brain phenotypes shown were highly penetrant, with approximately 75-100% of animals showing the growth phenotypes described. Images were analyzed in Zeiss Zen Software and processed in Photoshop. Larval Drosophila brain hemisphere volumes were analyzed using Imaris software. Larval glial cells were counted manually in representative optical sections of age-matched brain hemispheres, matched for section plane. Statistical analyses were done using Prism.