Ba4 mouse anti elastin antibody

Human dermal fibroblasts (GM3348; obtained from the Coriell Research Institute) cultured in DMEM with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin and human retinal pigmented epithelium cells (ARPE-19; obtained from Dr. M. Madigan, Save Sight Institute, New South Wales, Australia) cultured in DMEM/Nutrient mixture F-12 with 10% (v/v) fetal bovine serum, 2 mml-glutamine, and 1% (v/v) penicillin/streptomycin were seeded on glass coverslips at 18,400 cells/cm². At 10 and 14 days post-seeding, respectively, 20 μg/ml WT or D72A tropoelastin was added to the GM3348 and ARPE-19 cultures. At 1, 4, 7, and 10 days after tropoelastin addition, cells were fixed with 4% (w/v) paraformaldehyde for 20 min and quenched with 0.2 m glycine. The cells were incubated with 0.2% (v/v) Triton X-100 for 6 min, blocked with 5% (w/v) bovine serum albumin at 4 °C overnight, and stained with 1:500 BA4 mouse anti-elastin antibody for 1.5 h and 1:100 anti-mouse IgG-FITC antibody (Sigma) for 1 h. The coverslips were mounted onto glass slides with ProLong Gold anti-fade reagent with DAPI (Invitrogen).

Human dermal fibroblasts (5 × 104 cells) were seeded on glass cover slips in the wells of 12 well tissue culture plates in Fresh Media (FM) containing DMEM (Life Technologies) with 10% (v/v) fetal bovine serum (FBS; Life Technologies) and 1% (v/v) Pen/Strep (Sigma). Cells were cultured at 37 °C 5% CO₂ and the media was changed every 2–3 days. On Day 10 of culture 1 mg tropoelastin (filter sterilized; 10 mg/ml in phosphate buffered saline (PBS)) was added to each well and the cells were cultured for a further seven days, with media changes on days 13 and 15. Control cell samples with no tropoelastin addition were cultured for 17 days. At 1, 3 or 7 days post-tropoelastin addition the cultured cells were washed twice in PBS then fixed with 4% (w/v) paraformaldehyde for 20 min and quenched with 0.2 M glycine. The cells were incubated with 0.2% (v/v) Triton X-100 for 6 min, blocked with 5% (w/v) bovine serum albumin at 4 °C overnight, and stained with a 1:500 dilution of BA4 mouse anti-elastin antibody (Sigma) for 1.5 h and a 1:100 dilution of anti-mouse IgG-FITC antibody (Sigma) for 1 h. The coverslips were mounted onto glass slides with ProLong Gold anti-fade reagent with DAPI (Invitrogen). Slides were left to set for 24 h then analyzed using a confocal microscope.

Wells were coated with increasing concentrations of tropoelastin at 4 °C overnight and washed with PBS to remove unbound tropoelastin. Wells were blocked with 3% (w/v) BSA for 1 h. Bound tropoelastin was separately detected with one of three primary antibodies as follows: (a) 1:2000 BA4 mouse anti-elastin antibody (Sigma); (b) 1:500 rabbit anti-C-terminal antibody (custom-made by Biomatik); or (c) 1:5000 mouse anti-domain 6 antibody (custom-made by AbMart). Wells were washed and incubated with 1:5000 goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase for 1 h. Wells were visualized with ABTS solution (1.04 mg/ml 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid, 0.05% (v/v) H₂O₂, 10 mm CH₃COONa, 5 mm Na₂HPO₄) at 37 °C for 1 h, and absorbance readings were measured at 405 nm.