Rabbit anti cd31 pab

To improve detection sensitivity, the Scg3-Phage or VEGF-Phage was amplified in BLT7FLAG bacteria to label each phage particle with ~400 copies of FLAG tag [46 (link)]. Amplified phages were purified for in vivo ligand binding as above, followed by sequential intracardial perfusions with 40 mL PBS for 4 min, 20 mL of 4% PFA for 2 min and then 10 mL PBS for 1 min. Retinas were isolated, permeabilized, blocked in Solution A (5% goat serum and 1% Triton X-100 in PBS) overnight at 4 °C, and incubated with anti-FLAG M2 mAb (Sigma, St. Louis, MO, USA; #F1804; 1:200) and rabbit anti-CD31 pAb (Abcam, #28364; 1:50) in Solution A for 2 days at 4 °C. After washing, retinas were incubated with Alexa Fluor 594-conjugated anti-mouse IgG H&L (Cell Signaling, Danvers, MA, USA; #8890S; 1:1000) and Alexa Fluor 488-conjugated ani-rabbit IgG (H + L) (Cell Signaling, #4412S, 1:1000) in Solution A overnight at 4 °C. Retinas were washed, flat-mounted in 50% glycerol in PBS and viewed under a Keyence structured illumination fluorescence microscope (SIM; Osaka, Japan; Model BZ-X800).

Paraformaldehyde-fixed cryo-sections of the coronal brain were treated for 30 min in antigen-retrieval solution (Dako) prior to serum-free protein blocking (Dako Cytomation). Sections were then hybridized with various primary antibodies (overnight at 4 °C): mouse anti-human Aβ [residues 1–16, mAb clone 6E10 (1:100; Covance)], rat anti-GFAP pAb (1:1000; Sigma-Aldrich), and rabbit anti-CD31 pAb (1:50; Abcam). Hybridization with primary antibodies was followed by incubation with fluorophore-conjugated secondary antibodies (1 h at 37 °C; donkey anti-mouse, anti-rat, and anti-rabbit; 1:200; Jackson Immuno Research Laboratories) conjugated with Cy2, Cy3, and Cy5. Sections were mounted using ProLong Gold with DAPI (Molecular Probes, Life Technologies). Negative controls were processed using the same protocol with the omission of the primary antibody to assess non-specific labeling. A Carl Zeiss Axio Imager Z1 fluorescence microscope equipped with ApoTome (Carl Zeiss MicroImaging, Inc.) was used for microscopic analysis. AxioVision (release 4.6.3) software (Carl Zeiss) was used to process and analyze the images.