Research microscope dm ire2

A Leica Research Microscope DM IRE2 equipped with I3 S, N2.1 S, and Y5 filter systems (Leica Microsystems AG, Wetzlar, Germany) was used for phase-contrast and epifluorescence microscopy. Images (1388 × 1039 pixels) were acquired using a high-resolution CoolSNAP^TM CCD camera (Photometrics, Inc., Tucson, AZ, USA) under the control of a computer running Leica FW4000 software (Leica Microsystems AG, Wetzlar, Germany). Digital images were processed using Adobe Illustrator CC 2015 (Adobe Systems, Inc., San Jose, CA, USA). The quantifications of cells or puncta were performed using ImageJ (version 1.49, National Institute of Health, Bethesda, MA, USA) software with the cell counter plugin (National Institute of Health, Bethesda, MA, USA). Gel imaging was processed using the AlphaImager^TM HP system (www.alphainnotech.com).

An Olympus microscope BX53 was used to take the fluorescence images containing DAPI staining. A Leica research microscope (DM IRE2) (Leica Microsystems AG, Wetzlar, Germany) was used for taking fluorescence images in Figure 5 and Figure 6. Figure 3E,F were made by confocal microscopy (Leica TCS SP8 microscope and LAS X software, Leica Microsystems, ver. 2.0.2.15022) [81 (link)], using the facilities of the Advanced Neural Imaging Center of the Korean Brain Research Institute (KBRI). We used Adobe Systems Photoshop 7.0 software (Adobe, San Jose, CA, USA) for processing digital images.

Images (1,388 × 1,039 pixels) were captured using a Leica DM IRE2 research microscope equipped with I3 S, N2.1S, and Y5 filter systems (Leica Microsystems AG, Wetzlar, Germany) and a high-resolution CoolSNAP™ CCD camera (Photometrics, Munich, Germany) under the control of a computer using the Leica FW4000 program. The digital images were processed using Adobe Photoshop 7.0 software (Adobe, San Jose, CA, USA).

Electroporation was essentially performed as described by Ito et al.^{38 (link)}. In brief, postnatal day 1 (P1) ICR mice were anesthetized by hypothermia, and then skin and skull were pierced with a sharp glass microcapillary containing a solution of plasmid [pEGFP-Q74 (3 μg/μl), pEGFP-Q74 (3 μg/μl) + pDsRed2 (2 μg/μl), or pEGFP-Q74 (3 μg/μl) + pDsRed2-NAGK (2 μg/μl)] in Tris-EDTA buffer containing 0.5% Fast Green. DNA solution (2 µl) was injected into lateral ventricles (LVs), and injected brains were immediately electroporated with a tweezers-type electrode using an ECM 830 electroporator (BTX, USA). Five pulses of 100 V of 50 ms duration were administered with a 950 ms pause between pulses. Electroporated animals were placed on a hot pad at 37 °C for several minutes and then returned to their mother. Pups were anesthetized by hypothermia 2 days later, and brains were isolated, fixed for 2 h at RT in 4% (w/v) PFA, washed in PBS, and incorporated into agarose (5%) blocks, which were then sectioned (30 μm) with a vibratome (DTK-2000 Microslicer, Dosaka EM Co., Kyoto, Japan). Sections were mounted on slides and images were acquired using a Leica DM IRE2 Research Microscope (Leica Microsystems AG, Wetzlar, Germany). Experiments were approved beforehand by the Institutional Animal Care and Use Committee of the College of Medicine, Dongguk University.