A1r mp confocal

One-Photon fluorescence images were recorded on an FV 1200-IX83 confocal laser scanning microscope (PerkinElmer, USA) with excitation filter of 488 nm as well as emission filter of 600 nm. Two-photon fluorescence images were detected on an A1R MP confocal laser scanning microscope (Nikon, Japan) with 820 nm femtosecond-pulsed wavelength excitation and 600–700 nm emission. During the confocal experiment, all parameters were remained constant.

After fixation and dehydration, the obtained brains were coronally cryo-sectioned to 40 μm-thick slices using a microtome (HM550, Thermo / Life Technologies). The only staining is to show cell nuclei by counterstaining with DAPI (Cat. #10236276001, Roche), and all the GFP signals are natural origins without any signal amplification. All images were obtained using a Nikon’s A1R MP+ confocal microscope equipped with a fast high-resolution galvanometer scanner.
To count the labeled neuron number in the indicated brain regions of each mouse, the coronal brain slices at similar positions were observed with an interval of 160 μm (one from continuous 5 40 μm-thick slices), and the labeled neurons at the indicated brain regions were counted. To measure the labeling intensity of GFP positive neurons, three slices at similar positions from the indicated brain regions of the same brain were selected, and the GFP intensity was quantified by ImageJ software v1.60 (NIH, USA). By this method, the quantified value of mean GFP intensity of the labeled cells was shown as the arbitrary unit (AU), which was calculated as IntDen/Area (IntDen, Integrated Density of GFP-labeled neurons; area, the total area of GFP-labeled neurons).

Confocal microscopy images were acquired in a plane at a depth of approximately 30 μm from the coverslip. Images with 512 × 512 pixels, corresponding to 107 × 107 μm², were acquired at a fast rate of 30 frames per second to follow the short-time dynamics and at a slow rate, between 0.07 and 0.33 frames per second, depending on sample, to follow the long-time dynamics. Image series were acquired using a Nikon A1R-MP confocal scanning unit mounted on a Nikon Ti-U inverted microscope, with a 60 × Nikon Plan Apo oil immersion objective (NA=1.40). The pixel size at this magnification is 0.21 × 0.21 μm. The confocal images were acquired with the maximum pinhole size allowed by the microscope, corresponding to a pinhole diameter of 255 μm. Time series of 10⁴ images were acquired for 2–5 different volumes, depending on sample.