Examiner

4D DIC imaging and quantitative evaluation of division plane angles were performed as previously described using SIMI Biocell software (SIMI Reality Motion Systems) [56 (link)]. Embryos were dissected from young adult hermaphrodites incubated for 120 minutes in Dent`s buffer, 80 μM forskolin or respective solvents as control. Live images were taken with a Zeiss Axioplan 2e and a Zeiss Examiner. Z-stacks with spatial spacing of 1 μm were taken every 35 ms for 300 min. Confocal and fluorescent images were collected with Zeiss LSM5 and LSM510 Meta setups.

Experiments were performed in deep anesthesia. Embryos were prepared as described above for intravital epifluorescence microscopy. Analysis of YS-derived macrophage trafficking in real-time in vivo was performed in Cx₃cr1^Cre:Rosa26^mT/mG embryos (E9.5, E10.5) using an upright spinning disk confocal microscope (Examiner, Zeiss, Germany) equipped with a confocal scanner unit CSU-X1 (Yokogawa Electric Corporation, Japan) and a CCD camera (Evolve, Photometrics, USA). Images were acquired with a 20x/ 1.0 NA or a 63x/ 1.0 NA water immersion objective (Plan Apochromat, Zeiss, Germany) using a laser with an excitation wavelength of 488 nm for the detection of macrophages and a laser with an excitation wavelength of 561 nm for the detection of the tomato fluorescence signal of all other tissues. Imaging and image processing was performed using Slidebook 6.0.11 Software (3i, USA) and Fiji (NIH, USA)^{70 (link)}.

For the potentiostatically controlled direct force measurements, silica-particles were glued onto a Hellmanex-clean glass slide using NOA 63 and a micromanipulator attached to an optical microscope (Examiner, Zeiss, Oberkochen, Germany). For the preparation of colloidal probes, first a small drop of UV-curing NOA 63 glue was picked up with an etched tungsten wire, and placed on the substrate. A silica particle, dried from aqueous solution onto a clean glass slide, was placed onto the drop with a clean micromanipulator needle. The placed bead was cured for 1 min with UV light. For the force measurements, 30 force curves were performed in an aqueous solution (ionic strength of 0.1 mM and pH 4). The working electrode was the electrochemical gripper electrode, a Pt-wire was used as counter electrode and a chlorinated Ag/AgCl wire was used as a pseudo reference electrode. The electrochemical cell has been controlled by the same potentiostat also used for CV-measurements. The half-cell potential of the pseudo-reference was controlled against a calomel electrode (RE2, BASi Inc.) in an aqueous solution with ionic strength of 0.1 mM and pH 4. Force deflection curves were averaged and evaluated using a homemade procedure. Approach and retraction curves have been baseline corrected individually.

A diode laser (DL-473, Rapp Optoelectronic) was coupled to the epifluorescent port of the microscope (Zeiss Examiner, equipped with a 40X water immersion objective; Carl Zeiss Microscopy GmbH, Jena, Germany) via fiber optics. The field of illumination was targeted to the granule cell layer for stimulation of PV interneurons, and to the outer molecular layer for stimulation of SST interneurons. TTL-controlled light pulses of 2 ms duration and 1-7 mW intensity (a range within which saturation of the postsynaptic current amplitude was reached) were used to stimulate both PV and SST interneurons.