Plan apochromat

Live neurons grown on the coverslip that were transfected appropriately were transferred to the imaging chamber equipped with heating plate base (Live Cell Instrument, Seoul, Korea); filled with imaging solution (mM: 120 NaCl, 3 KCl, 2 CaCl₂, 2 MgCl₂, 15 glucose, 15 HEPES, pH 7.35), and imaged at 32 °C. Confocal images were acquired using the Revolution XD System (Andor Technology) equipped with Yokogawa CSU-X1 spinning disk confocal unit, 488 nm solid state laser, 561 nm solid state laser, 640 nm diode laser, and Andor 6-line laser combiner. Images were taken using a 60x (NA 1.4) or 100x Plan Apochromat objective (NA 1.4) and a 14-bit iXON3 DU-885 EMCCD camera (Andor Technology) using the Metamorph software program (Molecular Device Inc.). We acquired a complete confocal z-sectioning of the region of interest, followed by maximal intensity projection to produce a two-dimensional image using Metamorph.
For glycine stimulation, neurons were treated with 200 μM glycine in Mg²⁺-free imaging solution with 0.5 μM TTX, 1 μM strychnine, 20 μM bicuculline methiodide for 3−5 minutes. Then, neurons were returned to imaging solution with 0.5 μM TTX, 1 μM strychnine, and 20 μM bicuculline methiodide. Neurons at DIV 15−17 were used for imaging experiments.

For indirect immunofluorescence, cells were grown on glass coverslips and fixed with ice-cold methanol or paraformaldehyde for 10 min, then permeabilized and immune-stained with appropriate antibodies. Fluorescence images were acquired using Nikon TE2000-U with Metamorph software (Molecular Devices). Immunofluorescence staining of PtdIns(4)P and PtdIns(4,5)P₂ was performed following published methods30 (link)31 (link)57 (link). The specificity of the PtdIns(4)P and PtdIns(4,5)P₂ antibodies was verified by pre-absorbing the antibody with PolyPIPosomes containing 5% of PtdIns(4)P or PtdIns(4,5)P₂ (Echelon) for 1 h at room temperature before incubation with samples.
Three dimensional structured illumination microscopy was performed following standard protocol. Briefly, an ELYRA Super-resolution Microscopy system (Zeiss) equipped with an alpha ‘Plan-Apochromat' 100 × /1.46 Oil DIC oil immersion objective and an Andor iXon 885 EMCCD camera was used to acquire raw images following standard protocols. Sections were acquired at 0.125-mm z-steps. Colour channels were aligned using alignment parameter from control measurements with 0.5-μm diameter multispectral fluorescent beads (Zeiss). Structured illumination reconstruction and image processing were performed with the ZEN software package (Zeiss). Final image processing was done using Adobe Photoshop (Adobe).