VEGF receptor heterodimers I Nilsson et al The murine ES cell line R1 (Nagy et al, 1993) was routine cultured on a layer of mitomycin-C-arrested mouse embryonic ﬁbroblasts in ES medium: DMEM-Glutamax supplemented with 15% FBS, 25 mM HEPES pH 7.4, 1.2 mM sodium pyruvate, 0.12% monothiolglycerol and 1000 U/ml recombinant leukaemia inhibi- tory factor (LIF; Chemicon International, Temecula, CA) and passaged every second day. All medium components, except LIF, were from Invitrogen. Differentiation of ES cells in 2D or 3D cultures were done essentially as described (Li et al, 2008). Brieﬂy, on day 0, ES cells were resuspended in ES medium without LIF to induce differentia- tion. Cells were then aggregated in drops (1200 cells/drop) hanging from the lid of a non-adherent culture dish to form EBs. After 4 days, EBs were collected and seeded out individually on eight-well chamber glass slides (BD Biosciences, Franklin Lakes, NJ) for immunostaining or for proximity ligation, or in groups on 60 mm cell culture dishes for immunoprecipitation (ip) and western blot analyses. To score for 3D angiogenic sprouting, day 4 EBs were seeded into 12-well dishes, in groups of 8–10, on a layer of 0.6 ml of solidiﬁed collagen type I solution (Ham’s F12 medium [Invitrogen], 5 mM NaOH, 20 mM HEPES, 0.117% NaHCO3, 1% Glutamax-1 [Invitrogen] and 1.5 mg/ml collagen type I (Cohesion Technologies Inc, Palo Alto, CA)). Immediately thereafter, a second layer of collagen solution was added on top. After 3 h, 1.2 ml of medium with or without indicated growth factors was added. EBs were maintained under normal growth conditions or supplemented with human VEGFA (50 ng/ml PeproTech, Rocky Hill, NJ), or human, processed VEGFC (100 ng/ml; produced in-house). When indicated, neutralizing anti-mouse VEGFR3 (31C1, 15 mg/ml; Pytowski et al, 2005) or isotype-matched control (15 mg/ml; Southern Biotech; Birmingham, AL) were added to the cultures. Co-immunoprecipitation assay Cultures grown in 60 mm dishes (5 105/dish), treated or not with growth factors and neutralizing antibodies as indicated, were lysed in Nonidet P-40 (NP-40) lysis buffer: 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EGTA, 1 mM PMSF, 5 mg/ml aprotinin, 5 mg/ml leupeptin, 100 mM Na3VO4, and 1% NP-40. Lysates were cleared by centrifugation and incubated on ice for 2 h with 1 mg of VEGFR2 or VEGFR3 antibodies. For immunoprecipitation of VEGFR2 or VEGFR3 from human cells (e.g. HSaVEC), sc505 or sc321 antibodies (Santa Cruz Biotechnology Inc, Santa Cruz, CA) were used. The corresponding antibodies used for mouse cells (e.g. EBs) were AF644 for VEGFR2 or AF743 for VEGFR3 (R&D Systems, Minneapolis, MN). Immunoprecipitates were collected using Protein G Sepharose and subjected to SDS–PAGE, followed by transfer to Hybond-C extra membranes (Amersham Biosciences, Uppsala, Sweden). Membranes were incubated with antibodies directed against VEGFR2, VEGFR3 or phosphotyrosine (clone 4G10; Upstate Biotechnology Inc, Lake Placid, NY), followed by horse- radish perioxidase-conjugated secondary antibodies (Amersham Biosciences, Uppsala, Sweden). In parallel, total cell lysates were analysed by immunoblotting for VEGFR2 phosphorylation (pY1175; Cell Signaling Technology Inc, Beverly MA) and b-actin content (Santa Cruz Biotechnology). Immune reactivity was visualized using the enhanced chemiluminescence plus detection system (Amersham Biosciences, Uppsala, Sweden). Assays were repeated three times and representative results are shown. Immunoﬂuorescent staining of EBs EB cultures were washed twice in phosphate-buffered saline (PBS) and ﬁxed in 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature. After permeabilization in 0.1% Triton X-100 and block with TNB (PerkinElmer Life Science) or Tris-buffered saline complemented with 3% bovine serum albumin and 0.1% Tween 20, antibodies against CD31, LYVE1, NG2 (Chemicon), VE-cadherin (R&D Systems), VEGFR2 (AF644) or VEGFR3 (sc321 or AF7431; R&D Systems) were applied. All samples were treated with Hoechst 33342 to visualize nuclei. Stained EBs were mounted on glass slides in Fluoromount-G (Southern Biotechnology, Birmingham, AL) and analysed by a Nikon Eclipse E1000 microscope with a Nikon Eclipse DXM 1200 camera (Nikon, Tokyo, Japan) or an LSM 510 META confocal microscope (Carl Zeiss, Oberkochen, Germany). Quantiﬁcation of CD31-positive vascular area was done on 4 objective micrographs using the Easy Image Analysis 2000 software (Tekno Optik, Huddinge, Sweden). Five-10 EBs per condition were examined and the results are shown in arbitrary units±s.d. Quantiﬁcation of VEGFR2 and VEGFR3 expression along vascular sprouts in 3D EBs was performed on four EBs per condition and repeated three independent times. In situ proximity ligation assay (in situ PLA) Cultures, treated as indicated, were immediately ﬁxed in 4% PFA on ice for 30 min and thereafter subjected to in situ PLA using Duolink Detection kit (Olink Bioscience, Uppsala, Sweden) according to the manufacturer’s instructions for Duolink Blocking solution and Detection protocol. Brieﬂy, slides were blocked, incubated with antibodies directed against VEGFR2 (sc-6251) and VEGFR3 (sc321; Santa Cruz Biotechnology) and thereafter incubated with PLA probes, which are secondary antibodies (anti-mouse and anti- rabbit) conjugated to unique oligonucleotides. Note that different commercial batches of secondary antibodies varied slightly in their capacity to detect VEGFR2/-3 heterodimers so that the absolute but not relative numbers of receptor complexes were different (see Figure 2C compared with Figure 2E). When indicated, primary antibodies were converted to PLA probes by conjugation to 50thiolated oligonucleotides as described earlier (Soderberg et al, 2006). Brieﬂy, 50 mg monoclonal antibody (anti-VEGFR2, sc-6251 from Santa Cruz or anti-VEGFR3, MAB743 from R&D) were treated with 30-fold excess of sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxy- late), Pierce Biotechnology (Rockford, Il). Unreacted sulfo-SMCC was removed by gel ﬁltration. Freshly reduced thiolated oligonu- cleotides were added at a ratio of 1:4 (antibody:oligonucleotide) and coupled overnight. The PLA probes were then puriﬁed by gel ﬁltration, removing unreacted antibodies and oligonucleotides. Circularization and ligation of the oligonucleotides was followed by an ampliﬁcation step. The products were detected by a complementary ﬂuorescently labelled probe. Slides were mounted using Vectashield (Vector Laboratories Inc, Burlingame, CA) and evaluated using an LSM 510 META confocal microscope (Carl Zeiss). Z-stack micrographs taken with the 40 /63 objectives were obtained. The number of heterodimers, visualized as bright ﬂuorescent signals, was counted in 10–15 ﬁelds/well. n (number of wells) ¼ 6 for HSaVEC analyses, and n (number of individual EBs) ¼ 8 for EB analyses. Representative results are shown from experiments repeated at least three times. Cell images obtained were exported using the AxioVision software (Carl Zeiss) in TIF format for further analysis and determination of heterodimers/cell in Blob-Finder image analysis software (Version 2.5), which has been developed by the Centre for Image Analysis, Uppsala University. Quantiﬁcations are given as mean þ s.d. Statistical evaluation Statistical analyses were performed using Student t-test and P-values o0.05 were considered signiﬁcant. *Po0.05, **Po0.01, ***Po0.001. Supplementary data Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org). Acknowledgements This study was supported by grants to Lena Claesson-Welsh from the Swedish Cancer Foundation, the Swedish Research Council and the EU frame work program Lymphangiogenomics, LSHG.CT2004- 503573. Ola So¨derberg was supported by grants from the Wallenberg Foundation, by the EU FP6 project ‘ENLIGHT’, and by the Swedish Cancer Foundation. Johan Kreuger was supported by grants from the Swedish Cancer Foundation, the Swedish Research Council, and the Swedish Foundation for Strategic Research. Conﬂict of interest The authors declare that they have no conﬂict of interest. 1386 The EMBO Journal VOL 29 | NO 8 | 2010 & 2010 European Molecular Biology Organization