VEGF receptor heterodimers I Nilsson et al Figure 2 In situ PLA detection of VEGFR2/-3 heterodimers in intact HSaVECs. (A) Schematic outline of the in situ PLA strategy showing: (i) dimerized receptors (VEGFR2 in blue and VEGFR3 in grey) reacting with primary antibodies; (ii) close proximity of oligonucleotide-ligated secondary antibodies allows a rolling-circle ampliﬁcation (RCA); (iii) detection of the RCA product by a ﬂuorescently labelled probe. (B) Detection of heterodimers (in red) in HSaVECs treated with vehicle (–), VEGFA or VEGFC for 8 min on cells labelled with FITC-conjugated phalloidin (green). Inset in the VEGFC panel shows high magniﬁcation to clearly visualize the PLA spots representing heterodimers. Scale bar ¼ 10 mm. (C) Quantiﬁcation of VEGFR2/-3 heterodimers in HSaVECs treated with vehicle (–), VEGFA (A) or VEGFC (C) in cells preincubated or not with neutralizing antibodies blocking ligand binding to VEGFR2 or VEGFR3. n ¼ 6. (D) Quantiﬁcation of VEGFR2/-3 heterodimers in HSaVECs treated with different human VEGF isoforms (VEGFA121, 145, 165 or 189) or VEGFC for 8 min. n ¼ 6. (E) Quantiﬁcation of VEGFR2/-3 heterodimers in response to VEGFA, VEGFC, VEGFD or PDGFB. Growth factors are indicated as A (VEGFA), C (VEGFC), D (VEGFD) and P (PDGFB). n ¼ 6. Note that a different batch of PLA probes was used in this analysis compared with other panels in the ﬁgure (see Materials and methods). (F) Turnover of VEGFR2/-3 heterodimers in HSaVECs. Cells were treated with VEGFC for different time periods from 10 min to 24 h and samples were processed for detection of in situ PLA signals. n ¼ 6. Asterisks in panels C–F indicate the degree of signiﬁcance (**Po0.01, ***Po0.001). Antibodies against the intracellular domain were preferred, as they would not be disturbed by the ligand–receptor inter- action. This was followed by incubation with two sets of secondary antibodies conjugated with oligonucleotide, unique for each type of secondary antibody. Ligation of the oligonucleotides by a bridging probe in a proximity-depen- dent manner, allows a rolling-circle ampliﬁcation. Finally, & 2010 European Molecular Biology Organization this product is detected by complementary ﬂuorescent probes. As shown in Figure 2B, treatment of HSaVECs with VEGFC induced formation of heterodimers in situ, distributed on the plasma membrane as well as in the cytoplasm. The number of heterodimers/cell increased 100-fold from basal, in response to VEGFC treatment (see quantiﬁcation in Figure 2C). The EMBO Journal VOL 29 | NO 8 | 2010 1379