the animals returned to the rearing room to continue development. Early results suggested that higher single doses sometimes produced less effect than lower single doses, possibly indicating that at high doses the drug, which is dissolved in ” DMSO, precipitated out as it was injected into the aqueous hemolymph. For this reason, and because we were concerned that newly expressed FGFRs might overwhelm single drug injections, two or three 0.5 mg injections spaced 24 hr apart were used rather than a single, larger injection. We found no difference in phenotype between animals receiving 2 vs 3 injections. Immunocytochemistry Animals at various stages of metamorphic adult development were anesthetized by cooling on ice. Brains were dissected under insect saline solution at 100 mm. The final step in all protocols, also unless noted, was clearing the brains or sections for 15 min each first in 50% glycerol in water, then in 80% glycerol in water, and finally mounting on slides in 80% glycerol. For some preparations, glial cell nuclei also were labeled with the nucleic acid stains Syto 13 or Syto 59. Sections were washed in 10 mM Tris, then incubated in the Syto dye 1:10,000 in Tris for 60 min, washed in Tris, and mounted in H2O/glycerol.Fixed brains were washed in PBS, cryoprotected in 10, 20, and 30% sucrose in 0.1 M phosphate buffer, pH 7.4, at 4uC, flashfrozen in liquid propane, and cryosectioned at 20 mm. Sections were then processed according to the apoptosis detection kit instructions, using propidium iodide to counterstain nuclei. Western blot Antennal lobes of three female animals at stage 7 of adult development were removed and solubilized in Novex lithium dodecyl sulfate sample buffer containing protease-inhibitor and phosphatase-inhibitor cocktails. Solubilized samples were run on a Novex NuPAGE 4 12% Bis-Tris gel and transferred to a PVDF membrane as described previously. Blots were incubated for 1 hr at RT in blocking solution, then ON at 4uC in blocking solution with anti-pFGFR antibody. Blots were then washed in TBS-Tween and incubated 4 hr at RT in blocking solution plus horseradish peroxidase-conjugated goat antirabbit antibody. Blots were washed again and developed using the Opti-4CN kit. An additional blot to compare pFGFR labeling of antennal lobes treated with DMSO or buy PBTZ 169 PD173074 was done as described above using lobes and attached nerves from two animals for each treatment. Because immunocytochemistry suggested residual labeling in AL neuron cell bodies following PD173074 treatment, these cell body clusters were removed from the tissue prior to processing in order to assess solely the effect on glia. Glial FGFRs in Glia-Neuron Signaling Confocal microscopy and image processing Sections were viewed on a Nikon PCM 2000 or a Zeiss 510 Meta laser scanning confocal system using Simple 32 software or LSM software, respectively. Optical sections were acquired at 1- to 5-mm intervals through the depth of the antennal lobe and saved as three-dimensional stacks. To examine the ” localization of FGFRs, HSPGs, and Syto dyes to cellular subcompartments, we used a 406, oil immersion EC PLANNEOFLUAR, N.A. 1.3 lens. Vehicle controls were always imaged along with experimental brains and imaging parameters were always held constant when comparing between controls and experimental brains or across developmental stages. Confocal image stacks were projected and merged in false color using Confocal Assistant or the Zeiss LSM image browser, and then i