A11058

For immunofluorescence staining, antigen retrieval of deparaffinized tissue sections was performed by microwave on the middle for 20 min. After being blocked with 5% donkey serum (Solarbio, Beijing, China) for 1 h at room temperature, the sections were incubated with primary antibodies at 4°C overnight, followed by incubation with anti-rabbit Alexa Fluor 488 (A21206; Life Technologies, Carlsbad, USA) or anti-goat Alexa Fluor 594 (A11058; Life Technologies) conjugated secondary antibodies. DAPI (Life Technologies) staining was performed for nuclear counterstaining. Fluorescent images were visualized by using a laser-scanning confocal microscope system (OLYMPUS, Tokyo, Japan). The following primary antibodies were used: goat polyclonal anti
-MCP-1 antibody (sc-1785; Santa Cruz Biotechnology, Dallas, USA) and rabbit polyclonal anti-Firefly Luciferase antibody (ab21176; Abcam, Cambridge, UK).

Cells collected by centrifugation were spread onto polylysine‐coated slides by centrifugation (300 rpm for 5 min) with a cytospin (Shandon). Cells were then fixed at room temperature for 10 min in 2% (w/v) paraformaldehyde in PBS pH 7.2. Slides were stored at −80°C prior to use. Cells were permeabilized using 100 μl permeabilization buffer (0.5% Triton X‐100, 50 mM NaCl, 300 mM sucrose, 20 mM HEPES pH 7.5, 3 mM MgCl2) for 3 min at RT. Non‐specific antibody binding was blocked with 3% bovine serum albumin in PBS (w/v) (PBS‐BSA) for 30 min. The permeabilized cells were incubated with the primary antibodies for 3 h at 37°C. After washing, the cells were then incubated with secondary antibodies (A11001, A11058, Life Technologies; 1/400) for 20 min at 37°C. Nuclei were stained with DAPI (50 ng/ml) diluted in Vectashield (Abcys) for 10 min at RT. Samples were fixed in 4% paraformaldehyde for 5 min. The stained cells were observed either on an Axioplan 2 imaging microscope (Carl Zeiss) or examined through a Nikon inverted microscope attached to a laser confocal scanning system (Leica). All antibodies were tested in individual staining reactions for their specificity and performance. Controls without primary antibody were all negative.

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).

Paraffin-embedded lung and kidney tissue sections were deparaffinized, and then immersed in 0.2% Triton X-100 for 45 min as described previously (8 (link)). Following blocking with 10% donkey serum (ab7475, Abcam Inc, Cambridge, MA) in PBS for 1 h, slides were immunostained with rabbit anti-SP-A antibody, anti-TLR-4 (ab13556, Abcam Inc, Cambridge, MA), anti-TNFR1(sc-374186, Santa Cruz Biotechnology, Dallas, Texas). For cell IF analysis, the cells were fixed with 4% paraformaldehyde, and examined with anti-TLR-4(ab13556, Abcam Inc, Cambridge, MA), anti-TNFR1(sc-374186, Santa Cruz Biotechnology, Dallas, Texas) and anti-Megalin antibody (sc-16478, Santa Cruz Biotechnology, Dallas, Texas) to confirm the types of cultured cells and to determine the purity and quantity of proximal tubular epithelial cells. Slides were stained using Alexa 488 (ab150073, Abcam Inc, Cambridge, MA) and/or Alexa 594-conjugated secondary antibodies (A11058, Life Technologies, Eugene, OR) at room temperature for 1 h for fluorescence visualization of primary antibodies.