Immunostaining Drosophila Larval Brains

Drosophila larvae were dissected in PBS, and larval brains were fixed in 3.7% formaldehyde with PBS + 0.3% Triton-100 (PBT) for 15 min. The samples were processed for immunostaining as previously described^{54 (link)}. Confocal images were taken from LSM710 and brightness and contrast were adjusted by Photoshop CS5.1. Primary antibodies used in this paper were guinea pig anti-Dpn (1:1000; J. Skeath), mouse anti-Mira (1:50, F. Matsuzaki), rabbit anti-Mira (1:500, W. Chia), mouse anti-Grh (1:10, AH Brand), rat anti-CD8 (1:250, Invitrogen, Cat#: MCD0800), rabbit anti-GFP (1:500, Molecular Probes, Cat#: A21311), rat anti-Elav (1:10, DSHB, Cat#: Rat-Elav-7E8A10), mouse anti-Pros (1:10, DSHB, Cat#: Prospero (MR1A)), mouse anti-Dac (1:5, DSHB, Cat#: mAbdac2-3), mouse anti-α-tubulin (1:200, Sigma, Cat#: T6199), guinea pig anti-Asl (1:200, G Rogers), mouse anti-γ-Tub (1:200, Sigma, Cat#: T5326), mouse Acetylated-Tub (1:100, Sigma, Cat#: T7451), rat anti-Kr (1:100, EADC, Cat#: 574), rabbit anti-Pdm1 (1:500, W. Chia & X. Yang), rabbit anti-PH3 (1:200, Sigma, Cat#: 06‐570), mouse anti-Dlg (1:200, DSHB, Cat#: 4F3 anti-discs large), rabbit anti-Klumpfuss (1:200, X. Yang) and guinea pig anti-Chro-C (1:500, this study). DNA was labelled by ToPro-3 (1:5000, Invitrogen, Cat#: T3605).