Arf4 Regulates Dendritic Spine Development Figure 3. Overall structure and morphology of Arf4+/2 brains are not altered compared to WT controls. (A–P) Hematoxylin and eosin- stained paraffin brain sections from WT and Arf4+/2 mice. Representative images of the hippocampus (A, E), DG (B, F), CA1 (C, G), CA3 (D, H), cortex (I, J, M, N), thalamus (K, O), and cerebellum (L, P) from WT and Arf4+/2 mice are shown. doi:10.1371/journal.pone.0046340.g003 Genotyping of Arf4+/2 mice Genotype of Arf4+/2 mice was determined by using 2 parallel PCRs. The first pair of primers consisted of the forward primer I8.1 (59- AGCATATTCCCCTACTTAACTGTGTCTC-39) and the reverse primer I8.1 Rev (59-CAAAGGTGTTGCGGCA- CAGA-39), both of which are in intron 1. The second pair of primers consisted of P8 and RT416, the same pair used to characterize the ES cell line. PCR conditions were as described for identification of the gene trap insertion site. DNA was prepared from 0.5 cm of cut tail from 21-day-old mice and digested overnight with sodium chloride-tris-EDTA buffer, followed by deactivation with Proteinase K. PCR products were electropho- resed on 2% agarose gels and stained with ethidium bromide. Preparation of mouse brain tissues and Neuro-2A cell lysates Brains from WT or Arf4+/2 mice were collected after a 1 min transcardial perfusion with saline. The hippocampus and cortex were dissected from one hemibrain of each mouse and were homogenized with low detergent lysis buffer as previously described [34]. Samples were centrifuged at 35,000 rpm for 30 min at 4uC using a TLA 100.2 rotor of an Optima TL Ultracentrifuge (Beckman Coulter, Brea, CA) and the lysates were analyzed for Arf4 using western blot. Neuro-2A cells were lysed with low detergent buffer and centrifuged at 12,000 rpm for 10 min at 4uC, and supernatant was collected for western blot analysis. Rabbit anti-Arf4 was from Protein Tech Group (Chicago, IL), and rabbit anti-actin was from Sigma. Horseradish peroxi- dase-coupled anti-goat IgG was from Dako (Carpentaria, CA). Primary neuron preparation, transfection, and immunocytochemistry Mixed hippocampal and cortical neuron cultures were prepared from E18–19 mice and grown on poly-L-lysine-coated coverslips as reported [20]. Neurons were fixed in PBS containing 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 5% normal goat serum in PBS for 1 hour at room temperature. Neurons were incubated for one hour at room temperature with primary antibodies to goat HA (1:500, Novus Biologicals, Littleton, CO), rabbit Flag (1:500, Sigma), rabbit BDNF (1:100, Santa Cruz Biotechnology), or mouse MAP2 (1:500, Sigma). Secondary fluorophore-conjugated antibodies included donkey anti-goat Alexa488, donkey anti-goat Alexa594, donkey anti-goat Alexa647, donkey anti-mouse Alexa647, and donkey anti-rabbit Alexa594 (1:1000, Invitrogen). Image analysis and quantification For spine analysis of fluorescent cells, serial confocal images were taken with a BX60 BioRad Radiance 40X dry objective with a digital zoom factor of 2 for low magnification or of 4 for high PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e46340