Alexa fluor 488 conjugated goat anti rabbit igg a 11034

Flow cytometry was done as previously described on mature-stage IEs grown in type O negative blood (24 (link)). IEs were incubated for 30 min on ice with either 0.2 µg/ml purified IgG from a rabbit immunized with NTS-IT4var07DBLα1 or 0.1 µg/ml purified IgG from a rabbit immunized with NTS-HB3var03DBLα1 (both gifts from Alex Rowe, United Kingdom). Bound antibodies were detected by adding Alexa fluor 488-conjugated goat anti-rabbit IgG (A-11034; Molecular Probes) at 1/500. Samples were analyzed in an LSRII (Becton, Dickinson) with FlowJo V10 software (Tree Star Inc.).

Cells were seeded in 3.5 cm dishes (1×10⁶ cells/dish) containing collagen-I-coated coverslips, and were treated with doxycycline as described above. Immunofluorescence staining and image acquisition was performed as described previously [33 (link)]. Antibodies used were: anti-SLCO2A1 (HPA013742, Sigma-Aldrich, St. Louis, MO) as the primary antibody, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (A11034, Molecular probes-Thermo Fisher Scientific, Waltham, MS) as the secondary antibody.

The cells were seeded on coverslips, which were placed in the 6-well plate in advance. Following treatment with or without TGF-β (5 ng/ml) for 48 h, the cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 10 min, blocked with 1% bovine serum albumin for 1 h at room temperature and incubated with anti-E-cadherin and anti-vimentin antibody at 4°C overnight. Subsequently, the cells were rinsed thoroughly with PBS, and were incubated with Alexa Fluor 546-conjugated goat anti-rabbit IgG (A-11010) or Alexa Fluor 488-conjugated goat anti-rabbit IgG (A-11034) (Molecular Probes, Eugene, OR, USA) for 1 h at room temperature in the dark. 4′,6-Diamidino-2-phenylindole (Sigma-Aldrich) was used to stain the nuclei for 5 min at room temperature. Following mounting with the antifade mounting medium (Beyotime Institute of Biotechnology, Haimen, China), the cells were visualized by fluorescence microscopy (BX60; Olympus, Tokyo, Japan).