Mouse anti human vimentin antibody

Hepatocytes (0.2 × 10⁶ cells) alone or with MSC (0.2 × 10⁶ cells, ratio 1 : 1) were seeded on 12 mm coverslips in a 24-well plate in 1 mL of complete F12 medium. After 3 days, cells were washed with phosphate-buffered saline (PBS) and fixed with a 10% formalin solution (Sigma-Aldrich, Buchs, Switzerland) for 12 min. Cells were then permeabilized with Triton X-100 0.1% diluted in PBS for 15 min, and epitopes were blocked using 0.5% bovine serum albumin (BSA) for 30 min. Hepatocytes were stained with an anti-pig albumin antibody (Abcam) diluted to 1/200 and a secondary Alexa Fluor 555 goat anti-rabbit antibody (Life Technologies) diluted to 1/500. MSC were stained with a mouse anti-human vimentin antibody diluted to 1 : 50 (Dako, Glostrup, Denmark) and a secondary Alexa Fluor 488 goat anti-mouse antibody (Life Technologies). For 5-ethynyl-2′-deoxyuridine (Edu) staining, the Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher) was used following the manufacturer's instructions. Coverslips were mounted using VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Cambridgeshire, UK). Images were acquired using a fluorescence microscope and LAS V4.5 software (Leica Microsystems).

Pseudoislets after 6 days culture were formalin fixed and paraffin embedded using Histogel (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s recommendations. Four-micrometer sections were treated with 0.01 mol/l citrate for 15 min in a microwave to unmask epitopes. To avoid nonspecific binding, slides were incubated with 0.5% BSA for 30 min at room temperature.
For detection of MSC, sections were stained with mouse anti-human vimentin antibody, diluted 1:50 (Dako, Glostrup, Denmark), and with Alexa Fluor 488 goat anti-mouse antibody (Life Technologies, CA, USA). For detection of islet cells, sections were stained with guinea pig anti-porcine insulin, diluted 1:500 (Dako), donkey anti-guinea pig coumarin AMCA, diluted 1:300 (Jackson Immunoresearch, West Grove, PA, USA), rabbit anti-human glucagon antibody, diluted 1:100 (Merck Millipore, Darmstadt, Germany), and Alexa Fluor 555 donkey anti-rabbit antibody (Life Technologies). Microscopic images were acquired using a fluorescence microscope (Mirax Midi, Zeiss, Germany) and Pannoramic Viewer (3DHISTECH, Hungary), and confocal laser scanning microscopy was performed using LSM700 equipment (Zeiss).