Image station 4000r pro imaging system

This technique was performed mostly as previously described [25 (link)]. Blocking with 2% BSA-TBS was performed for 30 minutes at room temperature. Incubation with the primary antibody mouse anti-PCSK9 (Cayman Chemicals; Ann Arbor, MI; diluted 1:1000) was performed overnight at 4°C. After incubating with the HRP-labeled anti-mouse secondary antibody (Fisher Scientific; diluted 1:2,000) or avidin-HRP (Fisher Scientific; diluted 1:500) for 1 hour at room temperature, the SuperSignal West Pico Chemiluminescence Substrate (Pierce ThermoFisher) was used for detection. Several exposures ranging from 0.5 s to 30 minutes were made using a Kodak Image Station 4000R Pro Imaging System (Bend, OR) and the Carestream Molecular Imaging Software-Standard Edition-v.5.4.2.18893 (New Haven, CT).

This technique was performed essentially as previously described [21 ]. Blocking with 2% BSA-TBS or 5% non-fat dry milk-TTBS (depending on whether the antibody was biotinylated or not, respectively) was performed for 30 minutes at room temperature. Primary antibodies used were mouse anti-PCSK9 (Cayman; diluted 1:1000), rabbit anti-annexin A2 (diluted 1:250), mouse anti-A1AT (diluted 1 μg/mL), goat anti-APOH (diluted 1 μg/mL), goat anti-actin (used as internal control in some studies; diluted 1:250), and biotinylated sheep anti-PCSK9 antibody (R&D Systems; diluted 1:150). After incubating with HRP-labeled secondary antibodies (diluted 1:2,000) or avidin-HRP (diluted 1:500), depending on the primary antibody, immunoreactive proteins were detected using the SuperSignal West Pico Chemiluminescence Substrate. Several exposures ranging from 0.5 s to 30 minutes were made using a Kodak Image Station 4000R Pro Imaging System (Bend, OR) and the Carestream Molecular Imaging Software-Standard Edition-v.5.4.2.18893 (New Haven, CT). When needed, quantitation of the Western blot signals was performed using the Imaging software.