Immunolabeling of Drosophila Brains

Staged Drosophila brains were dissected in phosphate-buffered saline (PBS) at room temperature (RT). Brains were then fixed in 4% paraformaldehyde (PFA) + 4% sucrose in PBS (pH 7.4) with constant circular rotation for 30 min at room temperature (RT). The brains were next washed 3X in PBS. After washing, the brains were placed in blocking solution (1% bovine serum albumin (BSA) + 0.5% normal goat serum (NGS) in PBS + 0.2% Triton-X 100 (PBST)) for 1 h with constant rotation. Brains were incubated at 4 °C overnight with primary antibodies in blocking solution (0.2% BSA, 0.1% NGS in PBST), and then washed 3X in PBST for 20 min with constant rotation. Primary antibodies used: chicken anti-GFP (Abcam, ab13970; 1:1000), rat anti-Cheerio (1:1000)^{29 (link)}, rabbit anti-Repo (a kind gift from Dr. Benjamin Altenhein, University of Cologne, Germany, 1:1000), and rat anti-CadN (Developmental Studies Hybridoma Bank (DHSB); 1:50). After washing 3X in PBST for 20 min, brains were incubated with secondary antibodies in blocking solution for 2 h at RT, followed by 3X final washes with PBST and PBS for 20 mins^{101 (link)}. Secondary antibodies used: 488 goat anti-chicken (Invitrogen, A11039; 1:250), 546 goat anti-rat (Invitrogen, A11081; 1:250), and 568 goat anti-rabbit (Invitrogen, A11011; 1:250).