Xlumplanfl 0.95na

Animals were electroporated with DNA constructs at stage of 45–46 and screened for those with sparsely transfected and well-isolated cells. For imaging, animals were anesthetized with 0.01% MS-222 (Sigma) and were placed in a Sylgard chamber covered by a glass coverslip. Images were collected every 4 h before and after each visual experience session (dark or STVE). Two-photon z-series were collected at 1 μm steps with a 20× water immersion objective (Olympus XLUMPlanFL 0.95NA) at 3–4× scan zoom using a custom-built microscope modified from an Olympus FV300 system^{20 (link)}.
Complete dendritic arbors of each neuron were reconstructed using a semi-manual function in the Filament module of Imaris (Bitplane, US). Total dendritic length and TBTN were automatically calculated by the software. 3D Sholl analysis calculated the number of branches that intersect concentric circles at increasing distances from the cell soma, using a customized Matlab program with reconstructed filament data exported from Imaris. The dendritic structural data of the control group (Figs. 3–6) was a subset of a previously reported data set[20 (link)]. These neurons were collected and processed in parallel with the GluACTP-expressing experimental groups.

We used a MOM-type two-photon microscope (designed by W. Denk, MPImF, Heidelberg; purchased from Science Products/Sutter Instruments, Novato, CA, USA). Both design and procedures were described previously (Euler et al., 2009 (link)). The system was equipped with a mode-locked Ti:Sapphire laser (MaiTai-HP DeepSee, Newport Spectra-Physics, Germany) tuned to 927 nm, two detection channels for fluorescence imaging (red, HQ 622 BP36; green, D 535 BP 50, or 520 BP 39; AHF, Tübingen, Germany) and a 20x objective (XLUMPlanFL, 0.95 NA, Olympus, Hamburg, Germany). The red fluorescence channel was used to visualize the retinal structure using SR101 staining (see above), the green channel to target EGFP-labelled Igfbp5 RGCs.