510 upright confocal microscope

A small well was dotted with a very small amount of vacuum grease (Dow Corning) to stabilize the severed head of a fly. Heads were placed antennae-side down. Biotinylated 3K tetramethylrhodamine dextran (Invitrogen D7162) was applied to the severed neck connective. Dextran was prepared as a 10% w/v solution (10g in 100 ml) in phosphate buffered saline (PBS); 0.5 μl of this solution was used to label each brain. Fly heads were then immersed in Drosophila external saline85 (link) (in mM: 103 NaCl, 5 KCl, 5 Tris, 10 glucose, 26 NaHC03, 1 NaH2P04, 1.5 CaCl2, 4 MgCl2, osmolarity adjusted to 270–285 mOsm, and bubbled with 95% O2/5% C02 to pH 7.1–7.4) and left at room temperature for 30–90 minutes. Brains were then dissected in PBS and fixed for 20 minutes in a 4% paraformaldehyde solution (in PBS). The brains were then stained using the immunocytochemistry protocol described below. Confocal fluorescence microscopy was performed using a Zeiss 510 upright confocal microscope. Confocal Z-stacks were acquired at 1 μm interval using a 40X objective. Images were stitched together using the Pairwise Stitching PlugIn developed for ImageJ86 (link).