Femtotips 2 needles

Imaging was performed on a Visitron Systems Olympus IX81 microscope with a CSO-X1 spinning disk using a UPlanS APO 1.3 NA (Olympus) 60× objective. Embryos 1–2 hr old were manually dechorionated, aligned in heptane glue on 22 × 50 mm coverslips, and covered with a 1:1 mixture of Halocarbon oil 700 and Halocarbon oil 27) (Sigma). Imaging was performed with 400 ms exposure per slice, with five slices per stack and a constant room temperature of 22°C. Embryos were injected using an Eppendorf Inject Man NI 2 and Femtotips II needles (Eppendorf). The anti-DSpd-2 and anti-Dgt6 antibodies were suspended in injection buffer (100 mM HEPES, pH 7.4, and 50 mM KCl), centrifuged at 13,500 × g for 20 min, and injected at a concentration of either 6 mg/ml (anti-Dgt6 or anti-DSpd-2, high concentration) or 1 mg/ml (anti-DSpd-2, low concentration). For cold-treatment assays, single embryos were imaged until metaphase was reached, at which point they were placed in 50 mm ice-cold Petri dishes and covered with 4°C Halocarbon oil. Following 90 min on ice, embryos were reimaged, with 30 s typically expiring between removal from 4°C and the initiation of imaging. In some cases, embryos were removed following 75 min on ice, injected with antibody, and then placed on ice for another 15 min prior to imaging.

Drosophila embryos 1- to 2-h-old were harvested from 1- to 5-day-old adults. Embryos were manually dechorionated and mounted on 22×50 cm coverslips with heptane glue. The embryos were covered with 1:1 mixture of halocarbon oil 700 and halocarbon oil 27 (Sigma). Images were acquired with Visitron Systems Olympus IX81 microscope with a CSO-X1 spinning disk using a UPlanS APO 1.3 NA (Olympus) 60× objective. Images were acquired at 10 s intervals, in which five stacks 1 µm apart were taken with 400 ms exposure with 10% laser power for all genotypes, except embryos expressing Dgp71WD-GFP, where 20% laser power was used. His-tagged Wac, Dgt3^N, Dgt5^N, and Dgt6^C were buffer-exchanged with injection buffer (100 mM HEPES pH 7.4, 50 mM KCl) and concentrated with 30 kDa size-exclusion columns (Amicon). Protein concentration was measured by Bradford assay. Proteins were injected at 5 µg/µl. Embryos were injected using Eppendorf Inject Man NI 2 and Femtotips II needles (Eppendorf).