Rabbit anti ha antibody

Under deep anesthesia, ICR mice were fixed transcardially with 4% PFA in phosphate-buffered saline, pH 7.4. Fifty- or 100-μm-thick coronal sections were prepared with microslicer (VT1200S; Leica Biosystems). Sections were stained with Hoechst 33342 and mounted. Fluorescence images of 100-μm-thick sections were taken with a confocal laser-scanning microscope (TCS SP8; Leica Microsystems) using HC PL APO CS2 20×/0.75 NA multiple immersion lens (Leica Microsystems). Images were taken using the tile scan function of the confocal microscope. Fluorescence images of 50-μm-thick sections were taken using HC PL APO CS2 100×/1.40 NA oil immersion lens (Leica Microsystems). Basal dendrites of the cortical layer 2/3 pyramidal neurons were randomly sampled and captured. Image stacks were deconvolved using Huygens Essential version (Scientific Volume Imaging). Spines on tertiary dendrites were identified and counted in the 3D projection images with respect to each mouse. For immunohistochemical analysis, 50-μm-thick sections were prepared as described above, and immunostained with rabbit anti-HA antibody (Covance), followed by incubation with Alexa Fluor 488-conjugated anti-rabbit antibody (Invitrogen) and DAPI. Images were taken as described above.

Goat anti-human VE-cadherin polyclonal antibody, rabbit anti-human Flk-1 (VEGFR-2) polyclonal antibody, and rabbit anti-vav2 polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse anti-phosphotyrosine antibody was purchased from BD Bioscience (San Jose, CA). Mouse anti-HA antibody conjugated agarose beads and rabbit anti-HA antibody were purchased from Covance (Berkeley, CA). Anti-p120 catenin antibodies were purchased from Santa Cruz Biotechnology and BD Bioscience. Rabbit polyclonal anti-phospho-ERK1/2 (extracellular signal-regulated kinases 1/2) antibody was purchased from Cell Signaling (Beverly, MA). Specific inhibitors for VEGFR-2 tyrosine phosphorylation (SU1498) and Src (PP2) were obtained from Calbiochem (Indianapolis, IN). ON-TARGET plus SMART pool siRNA duplexes targeting the human P2Y₂ receptor, VE-cadherin and p120 were purchased from Dharmacon (Chicago, IL). VE-cadherin cDNA was a kind gift from Dr. Elisabetta Dejana (IFOM-IEO, Milan, Italy). All other reagents including nucleotides were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise specified.