Fv300 confocal microscope

Striatum slices (30 μm) prepared in a Leica 1000S vibratome (Nussloch, Germany) were incubated with the antibodies rabbit anti-myelin basic protein (MBP; 1:400, Abcam, Cambridge, UK), rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1; 1:1000, Wako Pure Chemical Industries, Japan), and rabbit anti-neuroglycan-2 (NG2; 1:200, Abcam, Cambridge, UK) as described [12 (link)]. After mounting the slices in Fluoroshield™ (Sigma-Aldrich, MO, USA), pictures were taken using a FV300 Olympus confocal microscope (Tokyo, Japan). For each animal and staining procedure, 3 sections were stained. In a separate analysis, three brain slices were directly stained at room temperature with 300 μL of green Fluoromyelin^TM (1:300 from the stock solution) (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min. Sections were rinsed, mounted in Fluoroshield™ (Sigma-Aldrich, MO, USA) and imaged using the same microscope. Quantification of fluorescence was performed as described [25 (link),26 (link)].

To detect the intracellular distribution of transduced Tat-HSP22 into HT-22, fluorescence microscopy was performed as described previously [24 (link)]. After exposure of HT-22 to Tat-HSP22 (5 μM) for 2 h, the cells were washed twice with PBS. Subsequently, the cells were staining with Histidine primary Ab and Alexa Fluor 488-conjugated secondary Ab. After washing, the cells were staining with DAPI (Roche, Mannheim, Germany) and observed under a FV-300 confocal microscope (Olympus, Tokyo, Japan).

SAGFF(LF)134A; Tg(UAS: GCaMP3) larval zebrafish were anaesthetized with Tricaine and mounted on a cover glass-bottomed culture dish in a drop of 1% LMP agarose in H₂O. The agarose applied to the anus was removed to expel the bolus. For the time-laps Ca²⁺ imaging of the smooth muscles (Figs. 2 and 5), we used a FV300 confocal microscope (Olympus) with a water emersion 20 × /0.75 or a water emersion 40 × /0.80 lens. Each z-projection image was obtained from 7 z-slices by 5 mm increment (Confocal aperture: 5) at 6 s interval, and the duration of each experiment ranged from 13 to 20 min. For the time-laps Ca²⁺ imaging of the larva expressing heat-shock induced GCaMP3 (Fig. 3), we used an SP8 confocal microscope (Leica) with a glycerol emersion 63x/1.30 lens. Each z-projection image was obtained from 16 z-slices by 5 mm increment at 12 s interval for about 8 min. Image post-processing was done with Image J.

The cyan and yellow fluorescent protein (CFP-YFP) based ratiometric Cl-Sensor protein (Markova et al., 2008 (link)) was delivered to CA3 pyramidal neurons in organotypic hippocampal slice cultures by biolistic transfection (Bio-Rad). At 2–3 d after transfection, Cl-Sensor protein expressing neurons were imaged using an FV300 confocal microscope (Olympus), custom-converted for multiphoton imaging, and equipped with a MaiTai-HP Ti:sapphire femtosecond pulsed laser (Newport Spectra-Physics). Images were acquired using Fluoview software (version 5.0, Olympus). Cells were excited at 850 nm and a 510 nm dichroic mirror was used to separate emitted light into CFP and YFP channels, which were filtered at 460–500 nm and 520–550 nm, respectively, and detected simultaneously using two externally mounted PMTs (Hamamatsu). Image stacks were flat-field corrected, collapsed along the z-plane, background subtracted, and the YFP/CFP ratio was calculated by dividing the respective images on a pixel-by-pixel basis. The ratio was calibrated to absolute intracellular chloride values using the K⁺/H⁺ exchanger nigericin and the Cl⁻/OH⁻ exchanger tributyltinchloride (both at 20 μm) in a high K⁺, HEPES-buffered solution at pH 7.35, as described previously (Boyarsky et al., 1988 (link); Kuner and Augustine, 2000 (link)).