Anti fibrillin 1 antibody

RASMCs/ Anu-RASMCs were treated in similar conditions as mentioned earlier. After 14 days, the cell layers were washed twice with PBS and fixed in 4% formaldehyde for 15 minutes in room temperature followed by incubation with a 5% bovine serum albumin blocking serum. The primary antibody, rabbit anti-rat elastin antibody (United States Biological, Swampscott, MA) or a rabbit polyclonal anti-fibrillin I antibody (Abcam, Cambridge, MA) at 1:100 dilution was applied overnight at 4°C. Alexafluor 488 chicken anti-rabbit IgG secondary antibody (Molecular Probes, Eugene, OR) was applied at a dilution of 8μg/ml for 2 hours at room temperature. Cell layers were mounted using aqueous mounting medium with anti-fading agents (Biomedia corp., Foster city, CA). The samples were examined by fluorescent microscopy. Importantly, all samples were imaged under exactly similar conditions for impartial analyses.

MOVAS cells were plated at 1×10^5 cells/ chamber in 2-chamber slides (Lab Tek II) and grown to the confluency for 3 days. Fibrillin was detected using anti-fibrillin I antibody (1:50 dilution, Abcam) with anti-rabbit IgG HRP secondary antibody (1:2000 dilution, Abcam) and DAB Peroxidase Substrate detection kit (Vector Labs) according to the manufacturer instructions. No counterstaining was used. Cells were imaged under the Nikon TE2000 microscope.