Anti periostin antibody

Anthocyanidin compounds Cy (purity ≥ 96%), Dp, Pg, Mv (purity ≥ 97%) and Pt (purity ≥ 95%) were from Extrasynthese (Lyon, France). Recombinant human TGF-β1 was from R&D Systems (Minneapolis, MN). Electrophoresis reagents were from Bio-Rad (Mississauga, ON). The anti-Twist1 monoclonal antibody was from Santa Cruz Biotechnologies (Dallas, TX). The anti-Periostin antibody was from Abcam (Cambridge, MA). Antibodies against Snail, phospho-Smad2, Smad2, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), vimentin, β-Catenin, phospho-Fak, Fak, phospho-Akt and Akt were from Cell Signaling Technology (Beverly, MA). The anti-fibronectin antibody was from Sigma Aldrich (Oakville, ON). Anti-mouse and anti-rabbit horseradish peroxidase (HRP)-linked secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA) and enhanced chemiluminescence (ECL) reagents were from Denville Scientific Inc. (Metuchen, NJ). Micro bicinchoninic acid protein assay reagents were from ThermoFisher Scientific (Rockford, IL).

A total of 96-well microplates were coated overnight with 1 μg/mL (0.1 μg per well) of anti-periostin antibody (R&D Systems, Minneapolis, MN). Plates were washed three times with 0.05% Tween 20 in PBS then blocked with reagent diluent for at least one hour. A total of 100 μL of all standards and patient samples was added to the 96-well plate and incubated for 2 hours. After 1-hour incubation with rabbit polyclonal anti-periostin antibody (Abcam, Cambridge, UK, 1:1000), 20 minute incubation with dextran polymer conjugated with horseradish peroxidase, and 20 minute incubation with substrate solution, stop solution was added to each well. Periostin absorbance was calculated by measuring at 450 nm, correcting for plate artifact at 570 nm using a log-transformed standard curve. Intra-patient variability of urine periostin was less than 12.5%.

SCC13 cells (1 × 10⁶ cells) were admixed with HDFs (5 × 10⁵ cells) with shRNA-mediated silencing of PDCD4 or control in 150 μl of growth factor-reduced matrigel (BD Bioscience) and intra-dermally injected into the back skin of 6-week-old NOD/SCID mice (Taconic Farms Inc.) as previously described [30 (link)]. For the ear injection, SCC13 cells (1 × 10⁵ cells) were admixed with equal numbers of HDFs with shRNA-mediated silencing of PDCD4 or control. Cells were resuspended in 3 μl of Hanks’ balanced salt solution and then injected intradermally into the left and right tip of the ear dermis of 10-week-old NOD/SCID mice using a Hamilton microsyringe fitted with 33 gauge needle as performed in [3 (link)]. Mice were sacrificed 3 weeks after injection and tumors were removed for analysis.
Tissue immunofluorescence was performed as before [2 (link), 3 (link)]. Anti- ki67 antibody, anti-Vimentin antibody (Abcam), anti-p63 antibody (Santa Cruz Biotechnology), anti-Involucrin antibody (Sigma), anti-Periostin antibody (Abcam), anti-Tenascin C antibody (Santa Cruz Biotechnology), and anti-αSMA antibody (Santa Cruz Biotechnology) were used. Quantification of all images of tissue immunofluorescence staining was performed using ImageJ and Adobe Photoshop software.
All animal studies were performed following the approved Institutional animal protocol procedure (IACUC-MGH).