Immunostaining of Drosophila CNS and PNS

For CNS immunostaining, whole flies immobilized with insect pin on abdomen were fixed in 4% PFA for 2 hours on nutator at room temperature. After three 1 min wash in PBT, flies were dissected in PBS buffer to get the whole CNS. CNS samples were further washed by three times in 1 min PBT and then blocked for 30 min in 4% NGS. Staining with primary antibody was performed in 4°C overnight on nutator. Samples were then washed 3 times for 20 min in PBT. Secondary antibody incubation was performed for 2 hours in room temperature. Samples were washed again in PBT for 3 times; mounted in VectaShield for imaging. PNS dissection and eye bristles immunostaining was performed using the published protocol [10 (link)]. In short, whole flies were washed in 100% ethanol and then PBS, specific body parts were then pulled and mounted in VectaShield on microscope slides for imaging. The following primary antibodies were used: chicken polyclonal to GFP (Abcam 13970, 1:500) and mouse monoclonal brp antibody (DSHB nc82, 1:200). The secondary antibodies were anti-chicken Alexa Fluor 488 (Invitrogen Molecular Probes A-11039, 1:500) and anti-mouse Alexa Fluor 633 (Invitrogen Molecular Probes A-21052, 1:500). Confocal images were taken on a Zeiss LSM710 microscope. Images were then processed in ImageJ.