Sc 17581

Cell or tissue samples were extracted with RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM ethylenediamine tetraacetic acid, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate, supplemented with protease inhibitors), and the total protein content was measured using the bicinchoninic acid protein assay kit (Thermo Scientific, Waltham, MA, USA). Proteins were resolved using denaturing SDS-PAGE followed by transferring onto nitrocellulose membrane. Primary antibodies against elastin (sc-17581), collagen (sc-8784), and actin (sc-8432) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Actin was used as the loading control. The immunoblot signal was detected using SuperSignal West Pico substrate (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Cell inserts were rinsed in PBS, fixed for 10 min in cold 4% PFA in PBS followed by fixation in 1% glutaraldehyde for 30 min at RT. The inserts were cut off with a razor blade and stored in PBS at 4 °C pending sectioning and immunohistochemical analyses. To analyze the bottom side of the inserts, they were placed face down on slides as flat mounts. For staining, slides containing flat mounts or vibratome sections were blocked with 1% BSA and incubated with primary antibodies anti C3/C3b/iC3b/C3d/C3dg (AB11862, Abcam), EFEMP1 (SDIX), MAC (C5b-9 AB55811, Abcam), TIMP-3 (AB39184 Abcam), ELN (sc17581, Santa Cruz Biotechnology), and CFH (AB53800, Abcam) overnight at 4 °C. Secondary antibodies labeled with Alexa-488 or Alexa-555 were incubated for 1 h at RT. Slides were mounted with Fluoromount G and visualized by TCS SP5 II confocal laser scanning microscope (Leica). Samples incubated with 1% BSA instead of primary antibody were used as negative controls.

Protein samples were extracted using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, pH 7.4, supplemented with protease inhibitors), and total protein concentration was determined with the Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, US). Proteins were resolved with SDS-PAGE, and subsequently transferred onto nitrocellulose membranes. Primary antibodies against elastin (sc-17581) and actin (sc-8432) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Actin was used as loading control. Immunoblot signal was detected using SuperSignal West Pico Substrate (Pierce, Rockford, IL, USA) following the manufacturer’s instructions.