Donkey anti goat hrp conjugated antibody

Cell culture supernatant was collected from TED orbital fibroblasts that were treated with vehicle, or treated with PPIs and/or 1 ng/ml TGFβ for 72 hours. A portion (5 μl) of culture supernatant was transferred to a PVDF membrane using a slot blot apparatus (Biometra, Gottingen, GE). The membrane was blocked with 5% nonfat dry milk in phosphate buffered saline with 0.1% Tween-20) and probed with a goat anti-collagen antibody (Santa Cruz Biotechnology, Santa Cruz, CA; cat. # sc-8783) overnight at 4°C. After washing, the blot was incubated with a donkey anti-goat-HRP conjugated antibody (Jackson Immunoresearch, West Grove, PA; cat. # 705-035-147) for 30 minutes. The membrane was developed with chemiluminescent substrate, and imaging was performed on an Azure c500 imager (Azure Biosystems, Dublin, CA). The densitometry of the bands was determined using Image Studio Digits, version 5.2 (LI-COR Biotechnology, Lincoln, NE). The RE was calculated by setting each intensity value of the vehicle treated sample to one.

To measure C5 present in mouse plasma, ELISA plates were coated with 1 μg/ml BB5.1 (anti-C5, catalog HM1073, Hycult Biotech via Cambridge Biosciences). After overnight incubation at 4°C, plates were blocked with 1% w/v BSA at room temperature for 1 hour and then washed. Plates were then incubated with diluted mouse plasma (1:1000) for 1 hour, washed, and then incubated with goat anti–human C5 (1:1000, Quidel A306, 073139). Plates were then incubated with donkey anti-goat HRP-conjugated antibody (1:5000, 705-036-147, lot 124700 Jackson ImmunoResearch Laboratories Inc. via Stratech Scientific). After a final wash, the plate was developed with TMB. Samples were standardized to WT control.

Anti-His₆ mouse monoclonal antibody (MAb) was purchased from GenScript (Piscataway, NJ). Anti-Penta-His MAb was purchased from Qiagen (Valencia, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody was purchased from Millipore (Temecula, CA). Isotype-specific goat anti-mouse antibodies were purchased from Sigma-Aldrich (St. Louis, MO). Adenovirus fiber antibody (4D2) was purchased from Abcam (Cambridge, MA). Donkey anti-goat HRP-conjugated antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).