2 M glycine (pH 2.4), neutralized by Tris-HCl (pH 8.6) after elution. Eluted proteins were

precipitated with 20% TCA overnight at 4°C, washed with cold acetone, resuspended in sample buffer, and analyzed by SDS-PAGE gel followed by Sypro-ruby stain. Appropriate bands present in the LRRTM4-Fc eluate but not in the Fc eluate from the 0.5 M NaCl elution were excised and analyzed by LC-MS mass spectrometry. All animal experiments were compliant with government and institutional guidelines. The targeting vector was generated by the recombineering method (Liu et al., 2003) from a 129S7 bMQ BAC clone, specifically bMQ30G17 from the Sanger Institute (Adams et al., 2005), and was www.selleckchem.com/btk.html confirmed by sequencing. The targeting vector contains a LoxP site and a neomycin cassette flanked by

Frt sites, replacing the major coding exon, Exon2, and a herpes simplex virus thymidine kinase expression cassette for negative selection. Targeting vector linearized by NotI was electroporated into 129/Ola embryonic stem (ES) cells for homologous recombination (Augustin et al., 1999 and Thomas and Capecchi, 1987). Positive selection of ES cells was in the presence of G418 and negative selection against random integration by Gancyclovir. Homologous recombination was verified by Southern blot analysis with a probe 5′ to the targeting vector. Positive ES cells were further expanded and a single Neo cassette insertion was verified by Southern blotting against a probe for the Neo cassette after two independent restriction digestions, by EcoRV and BglII. Positive clones were injected into C57BL/6J blastocysts, which were buy Epacadostat implanted into surrogate female mice. Founder chimeras were backcrossed six to seven generations with C57BL/6J mice. Genotypes were regularly ascertained by PCR analysis. Neurons, COS7 cells, and cocultures were fixed for 12–15 min with warm 4% formaldehyde and 4% sucrose in PBS (pH 7.4) followed by permeabilization with 0.25% Triton X-100, except where live staining or staining of unpermeabilized fixed already cultures was used. Live stainings were followed by fixation with warm 4% formaldehyde/4% sucrose

in PBS (pH 7.4). Fixed cultures were then blocked in 10% BSA in PBS for 30 min at 37°C and primary antibodies applied in 3% BSA in PBS. After overnight incubation at 4°C, the coverslips were washed with PBS and incubated in secondary antibodies in 3% BSA in PBS for 1 hr at 37°C. The coverslips were then washed and mounted in elvanol (Tris-HCl, glycerol, and polyvinyl alcohol, with 2% 1,4-diazabicyclo[2,2,2]octane). Immunofluorescence studies on coronal cryostat sections 20 μm thick at hippocampal level were performed on 6-week-old perfused LRRTM4−/− and littermate wild-type male mice. Fresh frozen sections were fixed by incubating in cold methanol for 10 min or for 7 min in 4% formaldehyde/4% sucrose and then blocked for 1 hr, followed by successive incubations with primary and secondary antibodies.