Journal: Brain Sciences

Article Title: Binge-like Prenatal Ethanol Exposure Causes Impaired Cellular Differentiation in the Embryonic Forebrain and Synaptic and Behavioral Defects in Adult Mice

doi: 10.3390/brainsci12060793

Figure Lengend Snippet: GDE enhances apoptosis and reduces proliferation in GD13.5 embryo forebrain tissues. Experimental design indicates developmental age, timing of ethanol exposure, and various analyses at early (GD13.5) and adult stages ( a ). Pregnant mice were exposed to 1.75 g/kg ethanol or saline i.p. at 0 h and again at 2 h on GD8 and GD12. Cleaved caspase-3 (CC3), a nucleoprotein expressed in proliferative cells (Ki67); ( b ) phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated cAMP-response element binding protein (pCREB), Paired Box 6 (Pax6), and LIM homeobox 2 (Lhx2); ( c ) levels were evaluated in forebrain tissues obtained from GD13.5 embryos by Western blot analysis. The protein samples were equally loaded, confirmed with Ponceau S staining, and normalized to β-actin or total proteins. Error bars, SEM (* p < 0.05 vs. GDS group; n = 8 embryos/group) (one-way ANOVA).

Article Snippet: The blots were incubated for 3 h at room temperature or overnight at 4 °C with the following individual primary antibodies: anti-rabbit cleaved caspase-3 (CC3) (Asp175) (polyclonal, #9661, 1:1000); anti-mouse-β-actin (monoclonal, #3700, 1:1000); anti-rabbit CREB (monoclonal, #9197, 1:1000); anti-rabbit phospho-CREB (monoclonal, #9198, 1:1000); anti-rabbit p44/42 MAPK (ERK1/2) (polyclonal, # 9102, 1:2000); anti-rabbit-phospho-p44/42 MAPK (polyclonal, # 9101, 1:1000); anti-rabbit GluA1 (monoclonal, #13185) from Cell Signaling (Danvers, MA, USA); anti-mouse-Arc (C-7, #sc-17839, 1:1000); anti-mouse-Egr1 (monoclonal, S-25, #sc-101033, 1:500) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); anti-rabbit nucleoprotein expressed in proliferative cells (Ki67) (polyclonal, #PA5-19462); anti-mouse LIM homeobox 2 (Lhx2) (monoclonal, # MA5-15834) from Thermo Fisher (Waltham, MA, USA); anti-rabbit Paired Box 6 (Pax6) (polyclonal, #12323-1-AP) from Proteintech (Rosemont, IL, USA); anti-rabbit fibroblast growth factor 1 (Fgf1) (polyclonal, #ab207321) from Abcam (Waltham, MA, USA); and anti-rabbit GluN1 (polyclonal, #05-432) from Millipore Sigma (Burlington, MA, USA).

Techniques: Saline, Binding Assay, Western Blot, Staining