Alexa fluor 488 and 594 secondary antibodies

Paraffin blocks of villous tissue were sectioned (5 μm thick) and collected on Excell Slides (Fisher Scientific, Pittsburgh, PA, USA). The slides were deparaffinised, followed by 30 min incubation in 10 mmol/l sodium citrate with 0.05% (vol./vol.) Tween solution (pH 6.0) in a 100°C water bath for antigen retrieval. The tissue sections were blocked for non-specific staining with PBS containing 0.3% (vol./vol.) Triton X-100 (Sigma-Aldridge, St Louis, MO, USA), 5% (vol./vol.) Normal Donkey Serum, 1% (vol./vol.) IgG-free BSA (Jackson Immunoresearch, West Grove, PA, USA) for 30 min. The slides with isolated cells or villous tissue were incubated overnight at 4°C with the following antibodies: TLR4 (H-80, 1:50; Santa Cruz, Dallas, TX, USA) and CD34 (ab8536, 1:400; Abcam, Cambridge, MA, USA). For visualisation, Alexa Fluor 488 and 594 secondary antibodies (1:1000; Jackson Immunoresearch, West Grove, PA, USA) were used. The nucleus was labelled with To-Pro 3 (Sigma-Aldridge). The slides were imaged with Leica Microsystem SP2 Confocal Microscope (Leica Microsystems, Buffalo Grove, IL, USA). All antibodies were diluted in PBS and validated by negative control.