Pm036

Eight fetal brains ranging from 29 to 34 WG were subdivided in two groups. Four brains belonging to the control group were obtained from fetuses whose brain was macroscopically and microscopically free of detectable abnormalities. Four brains from pFAS/FAS fetuses were obtained after spontaneous in utero death (Supplementary Tables 1 and 2).
Seven micrometer tissue sections from the frontal cortex were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Antigen retrieval was performed by heating the sections in 10 mM boiling sodium citrate buffer (pH 6.0) for 15 min. Then, sections were incubated overnight at 4 °C with anti-GLUT1; 1/200 (sc-1605, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-LC3; 1/200 (PM036, MBL International Corporation, Woburn, MA, USA) as previously described.^{17 (link)} Afterwards, sections were incubated with fluorescent-conjugated antibodies (Alexa Fluor 594-conjugated donkey anti-goat; 1/400 (A11058, Life Technologies, Waltham, MA, USA), Alexa Fluor 488-conjugated donkey anti-rabbit; 1/400 (A21206, Life Technologies)). Confocal images were acquired with the Leica laser scanning confocal microscope TCS SP2 AOBS (Leica Microsystems, Wetzlar, Germany).

Endothelial cells or brain cortical microvessels were washed three times with PBS and fixed by incubation in 4% PFA in PBS for 1 h at room temperature. After incubation with 1% BSA/0.3% Triton X-100 in PBS to block nonspecific sites, primary antibodies (anti-LC3; 1/200 (PM036, MBL International Corporation), anti-GLUT1; 1/200 (sc-1605, Santa Cruz Biotechnology)) were added, and the preparations were incubated overnight at 4 °C. Secondary antibodies in blocking solution were then incubated with the preparations for 1 h at room temperature (Alexa Fluor 594-conjugated donkey anti-goat; 1/400 (A11058, Life Technologies), Alexa Fluor 488-conjugated donkey anti-rabbit; 1/400 (A21206 Life Technologies)).