Bx51wi dsu confocal microscope

On PND 15, mice were transcardially perfused with ice-cold 0.9% saline followed by ice-cold 4% paraformaldehyde in phosphate buffer (PB), pH 7.4. Brains were removed, postfixed at 4°C and successively immersed in 20% and 30% sucrose cryoprotection solutions. Sections (22 μm) were collected in 24-well culture plates filled with 0.9% phosphate buffered saline (PBS), pH 7.4. After 3 washes with PBS + 0.3% Triton X-100 (PBST), the sections were incubated overnight at 4°C in rabbit anti-xCT (1:100) or rabbit anti-EAAC1 (1:300) and chicken anti-MAP2 (1:800) primary antibodies with a 2% normal horse serum in PBST solution. After 3 washes in PB solution sections were incubated with anti-rabbit Alexa Fluor 594 and anti-chicken Alexa 488 secondary antibodies (1:300) diluted in PB solution for 2 h. Finally, sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and analyzed under the microscope. Photomicrographs were acquired with an Olympus BX51WI DSU confocal microscope (Olympus, Center Valley, PA, USA) coupled to a Hamamatsu EM-CCD C9100 camera (Hamamatsu, Hamamatsu, Japan).

For all imaging experiments on (non-cleared) vibratome sections an Olympus BX51WI DSU confocal microscope (Olympus, Center Valley, PA, USA) coupled to a Hamamatsu EM-CCD C9100 camera (Hamamatsu Photonics K. K., Hamamatsu, Japan) was used. The system was equipped with a motorized stage and a LEP MAC 5000 Controller System (Ludl electronic products, Hawthorne, NY, USA). Images were taken with either, an Olympus PlanApo 2x/0.08 NA, 4x/0.16 NA or UPlanSApo 10x/0.40 NA objective (Olympus, Center Valley, PA, USA). Excitation and emission characteristics for all dyes are given in Supplementary Table 2. For acquisition the Stereo Investigator software (MBF Bioscience, Williston, Vermont, USA) was used.