Tsc sp5 2

At day 21 of culture, live cells were visualized by a vital staining with fluorescein diacetate (FDA) on all experimental groups as described previously [17 (link)]. Briefly, 5 mg/mL FDA stock solution (Invitrogen) was prepared in acetone: 40 μL stock solution was diluted in 10 mL phosphate buffer solution (PBS) (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM NaH₂PO₄, pH 7.4) and 250 μL was mixed with 500 μL culture medium. Cells were incubated with a working solution for 10 min. The live cells were examined by a confocal laser scanning microscope model TSC SP5 II (Leica Microsystems, Bensheim, Germany), using a 40× oil immersion objective.

Mice were deeply anesthetized and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, PB). The fixed brains were cryoprotected and sectioned into 40-μm-thickness slices using the freezing microtome. Sections of 20-μm-thickness were prepared by using a cryostat (CM1850, Leica Microsystems) and mounted on MAS-coated glass slides (Matsunami Glass). Sections were immunolabeled with primary antibodies against calbindin (AB1778, Chemicon or #300, Swant), phospho-S6 (#2211, Cell Signaling Technology), cleaved caspase-3 (#9661, Cell Signaling Technology), HIF-1α (ab1, Abcam), heme oxygenase-1 (ab13248, Abcam) and VGluT2 (kind gifted from Prof. Masahiko Watanabe at Hokkaido University). Bound antibodies were visualized with Alexa-488 or Alexa-633 conjugated-secondary antibody (Life Technologies) and TO-PRO-3 were used for nuclear staining (Life Technologies). The sections were coverslipped with Vectashield Mounting Medium (H-1000, Vector Laboratories), and viewed by a confocal laser scanning microscope (TSC-SP5II, Leica Microsystems). For cytochrome oxidase histochemistry, sections were washed with PBS, and incubated for 4 h at 37 °C in a solution containing cytochrome c (3 mg, Sigma), 3,3-diaminobenzidine (5 mg, Sigma), and sucrose (450 mg) in 10 ml of 0.1 M PB.