Bsa pbs

Microtiter plates were coated with 5 μg/well of Col I, III, IV, and V (Sigma) in bicarbonate buffer, pH 9.6, and incubated overnight at 4 °C. After blockade with 1% BSA-PBS (Sigma) for 60 min, duplicates of the mouse serum samples (1:100) were diluted in 1% BSA-PBS with 0.05% Tween 20 and incubated for 60 min at room temperature, followed by incubation with goat-anti-mouse IgG conjugated alkaline phosphatase (Sigma). The reaction was revealed by adding 1 mg/ml of p-nitrophenyl phosphate (pNPP) as substrate diluted in 1 M diethanolamine and 0.5 mM MgCl₂ in a pH 9.8 buffer. The optical density (OD) of the samples were read at 405 nm on a microplate reader (Labsystems Multiskan). The cutoff was determined based on the means plus 3 SD (standard deviation) of eight normal serum samples.

Protein validation was performed by immunohistochemistry on 7 µm thick frozen sections of non-decellularized tissue. Frozen sections were used for a standard hematoxylin and eosin staining and for a protein-specific staining using fluorescent secondary antibodies. Acetone-fixed sections were blocked with 1% BSA in PBS for 1 h. Polyclonal primary antibodies against EMILIN1 and FBN1 were diluted in 1% BSA/PBS (1:100 and 1:200, respectively; both Sigma Aldrich, Zwijndrecht, The Netherlands) and incubated for 1 h at room temperature. Sections were washed 3 times in PBS/T solution and incubated with Alexa Fluor 488 donkey anti-goat IgG (1:100; Life Technologies) and αSMA-Cy3 (1:500; Sigma) diluted in 1% BSA/PBS for 1 h at room temperature. After 3 times washing with PBS/T solution, sections were incubated with DAPI for 5 min, washed with PBS, and mounted. Stained sections were imaged using fluorescent microscopy (Olympus IX53, Olympus Leiderdorp, The Netherlands) and ImageJ (version 1.47) to analyze EMILIN1, FBN1, and αSMA positive area.

F-actin and vimentin staining was performed according to previously described protocol [59 (link)]. In short, A549 cells grown on sterile glass coverslips were fixed with 4% paraformaldehyde (Serva, Heidelberg, Germany) and permeabilized with 0.25% Triton X-100. F-actin was labeled with phalloidin conjugated to tetramethylrhodamine isothiocyanate (TRITC diluted 1:5 in PBS, 20 min; Sigma-Aldrich, St. Louis, MO, USA). Non-specific background was blocked with 1% bovine serum albumin (BSA). Mouse monoclonal anti-vimentin antibody (diluted 1:50 in BSA-PBS, 60 min; Sigma-Aldrich, St. Louis, MO, USA) was used as a primary antibody to label vimentin (diluted 1:50 in BSA-PBS, 60 min; Sigma-Aldrich, St. Louis, MO, USA), followed by the incubation with a secondary goat anti-mouse antibody conjugated to Alexa Fluor 488 (diluted 1:100 in PBS, 60 min; Sigma-Aldrich, St. Louis, MO, USA). Cell nuclei (DNA) were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St. Louis, MO, USA). All of the incubations were done at ambient temperature, whereas fluorescent staining was performed in the dark. Finally, slides were mounted in Aqua-Poly/Mount (Polysciences, Warrington, PA, USA) and examined using a Nikon Eclipse E800 fluorescence microscope and NIS-Elements 4.0 software (both from Nikon, Tokyo, Japan).