to remove axons and myelin fragments from the vascular pre- paration. Pelleted total segments were collected in divalent cation free phosphate buffered saline with 0.1% bovine serum albumin (BSA-PBS, Sigma, St. Louis, MO, USA) and selectively screened by size [16]. After normal microvessel fragment isolation, the fragments of arterial identity containing magnetic beads in their lumens were separated from venules and capillaries by the use of a magnet (DynalTM, Invitrogen, Carlsbad, CA, USA). Isolated aMFs were repeatedly (5X) exposed to the magnet and washed to improve recovery and enrichment. Microvascular construct preparation and implantation After isolation of microvessel segments from rodent brain cortex or adipose tissue [16,46] isolated vessel segments were suspended into liquid collagen type I at a density of 20,000 segments/ml. The collagen was prepared by mixing sterile, acidified rat tail collagen Type I (BD Biosciences, San Jose, CA, USA) with 1/4 volume of ice cold 4x Dulbecco’s Modified Eagle’s Media (DMEM) (Invitrogen, Carlsbad, CA, USA) and 1 M NaOH to yield a final concentration of 3 mg/ml collagen. Segment/collagen suspen- sions were pipetted into wells of a 48-well culture plate (0.2 ml/ well) and incubated at 37uC to polymerize the collagen to form a three-dimensional microvascular construct (MVC). MVCs were either cultured in DMEM+10%FBS or implanted subcutaneously for 1 or 6 weeks on the flanks of immunodeficient mice (B6;129S7- Rag1tm1Mom/J) according to published methods [17,46]. Mice were anesthetized with isoflurane (initially 4–5% in an induction chamber then 2% via intubation tube) via precise vaporizer. Monitoring of adequate anesthesia is done by toe pinchs. A sub- cutaneous pocket was formed between the skin and the underlying muscle anterior using blunt dissection through a small skin in- cision. Each host mouse received 2 implants. The incision was then closed with surgical staples and the animal was allowed to recover. Unless indicated otherwise, microvessel isolates were prepared from the GFP transgenic rats. Implanted Microvascular Construct Visualization MVCs were harvested at different time points following implantation. For that, mice were anesthetized with isoflurane (4–5% in an induction chamber) then euthanized by cervical dislocation. MVCs were fixed in 4% paraformaldehyde for 2 hours and permeabilized with 0.5% Triton X-100 and rinsed with PBS. After blocking for 2vhours with 10% goat serum (Sigma, St. Louis, MO, USA), samples were incubated overnight with different antibodies or lectins: Anti-EphB4 (R&D systems), biotinylated Griffonia simplicifolia I (GSI; Vector labs, Burlin- game, CA, USA), GSI-TRITC, streptavidin-Cy3 (Vector labs), streptavidin-Cy5 (Vector labs), anti-a-smooth muscle actin (Sig- ma), Zenon kit Alexa Fluor-594 (Invitrogen). Samples were imaged en bloc with an Olympus MPE FV1000 Confocal Micro- scope and resulting images analyzed with Amira 5.2 (Visage Imaging, Inc, CA). Vessels were identified by either endogenous expression of GFP or stained with GSI lectin. To identify functional microvessels in the implanted constructs, host mice were injected intravenously with a bolus of Dextran-TRITC 2,000,000 MW (Sigma, St. Louis, MO, USA) as a blood tracer 15 min before the constructs were harvested. As previously described [16], implanted vessel fragments persist and form a vessel network without significant contribution of host cells. To detect LacZ activity, MVCs were harvested, fixed as described above and cryoprotected in 30% sucrose in PBS at 4uC for 3 days followed by mounting in freezing medium (Tissue-Tek O.C.T. Compound, Sakura Finetek, Torrance, CA). MVCs were sectioned in 20 mm slices utilizing a LEICA CM3050 cryostat Arterial-Venous Plasticity in Adult (Meyer Instruments, Inc, Houston, TX), thawed at room tem- perature, washed 3 times in PBS, fixed with freshly prepared 0.5% glutaraldehyde in PBS, washed 3 times for 15 min in wash buffer (0.1 M PBS pH 7.3, 2 mM MgCl2, 0.02% NP-40) and incubated with 1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-b-galactopyra- noside, Sigma, St. Louis, MO) in wash buffer containing 5 mM potassium ferrocyanide and 5 mM potassium ferricyanide until desired staining level was achieved. Implanted MVC Analysis After volume reconstruction from image stacks using Amira 5.2 (Visage Imaging, Inc, CA), vessel diameter and % vessel type was measured for each microvessel element present in a field of view. The fraction of the vascular volume comprised of arterial identity was quantified by determining the ratio of the vascular volume positive for GFP (i.e. EphrinB2-reporter) to the total vascular volume (GSI-positive). Three fields of view per MVC were counted per 3 different implanted MVCs. Protein quantification by Western blotting MVCs were cultured in vitro for 7 days in DMEM containing 10% FBS to generate angiogenic neovessels. RIPA buffer (20 mM TRIS- HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/ml leupeptin, Cell Signaling, Danvers, MA) containing protease and phosphatase inhibitors (HaltTM protease and phosphatase inhibitor cocktails, Thermo Scientific) was added to either fresh isolates (mature specified vessels) or from angiogenic MVCs. Samples were ho- mogenized on ice with a glass homogenizer to macerate the collagen and incubated on ice for 30 min with mild agitation before centrifugation at 14,000 rpm, 4uC for 10 min. Supernatant was harvested and utilized for Western blotting following 4–15% gradient SDS-PAGE (BioRad, Hercules, CA). EphrinB2 was detected with a biotinylated anti-EphrinB2 antibody (Leinco, St. Louis, MO) followed by streptavidin-HRP (DAKO). The bands were detected with chemifluorescence substrate ECL plus (Amer- sham, GE healthcare) with a Typhoon 9400 gel imager (GE Healthcare, Piscataway, NJ) and were quantified by densitometry using MetamorphTM imaging software (Molecular Devices, Inc). Analysis of EphrinB2 phosphorylation by Western blotting Lysates obtained as described above were also used for EphrinB2 immunoprecipitation. However, 1X volumes and 5X volumes of lysates were used for immunoprecipitation from freshly isolated vessels and angiogenic MVCs, respectively. Lysates were incubated with anti- EphrinB2 antibody (Novus Bio, Littleton, CO) and protein A/G agarose beads (Pierce, Rockford, IL) overnight at 4uC. Beads were washed 5X with RIPA buffer containing protease and phosphatase inhibitors, harvested and utilized for Western blotting following 4– 15% gradient SDS-PAGE (BioRad, Hercules, CA). EphrinB2 was detected with a biotinylated anti-EphrinB2 antibody (Leinco, St. Louis, MO) followed by strepavidin-HRP (DAKO). EphrinB2 tyrosine phosphorylation was detected with anti-P-Tyr (Millipore, Billerica, MA). Bands were detected and quantified as above. Statistical analysis Data are expressed as mean 6 s.e.m. p-values were calculated using Student’s t-test (SigmaStat v3, Systat software) for all measurement comparisons except for vessel diameter distributions which involved using Kruskal–Wallis One Way Analysis of Variance on Ranks. p,0.05 was considered statistically significant. PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27332