Pierce fast blocking buffer

Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane. After treatment with Pierce Fast Blocking Buffer (Pierce Biotechnology Inc., Tokyo, Japan), the membrane was incubated with buffer containing mAb 4713 (1 μg/mL), followed by a horseradish peroxidase-conjugated secondary antibody. The membrane was treated with enhanced chemiluminescent (ECL) reagent (Amersham; Tokyo, Japan), and the reactive protein bands were visualized with a Fujifilm image analyzer.

MSTO-211H, Huh-7 cells, and transfected Huh-7 cells (3 × 10⁶ cells/well) incubated, with or without rfhSP-D (20 μg/mL), in a serum-free Roswell Park Memorial Institute (RPMI) medium for 12 and 24 hours. The cells were lysed in a lysis buffer (50 mM Tris-HCL pH 7.5, 150 mM NaCl, 1% Triton X, 1 mM sodium orthovanadate, 10 mM β-glycerophosphate, 2 mM ethylenediaminetetraacetic acid (EDTA), and 10 mM sodium pyrophosphate) and analyzed by western blotting. Lysate proteins (30 μg) were separated by 15% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto poly vinylidene di fluoride (PVDF) membranes (Pall Corporation, NY, USA). Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane. After treatment with Pierce Fast Blocking Buffer (Pierce Biotechnology, Inc., Tokyo, Japan), the membranes were incubated with a buffer containing JMAM-1 mAb (1 μg/mL), followed by a horseradish peroxidase-conjugated secondary antibody. The membrane was treated with an enhanced chemiluminescent reagent (Amersham, Tokyo, Japan), and the reactive protein bands were visualized using a Fujifilm image analyzer.

Immunofluorescence microscopy was performed following the method of Hiong et al. (2017b (link)). The concentrations of anti-DDCA antibody and secondary antibody Alexa Flour 488 used were 2.5 μg ml⁻¹. Both primary and secondary antibodies were diluted in Pierce Fast Blocking Buffer (Thermo Fisher Scientific Inc.). A peptide competition assay was also performed to confirm the specificity of the anti-DDCA antibody following the methods of Hiong et al. (2017b (link)). The slides were observed using Olympus BX60 epifluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with Olympus DP73 digital camera (Olympus Corporation). Figures were prepared using Adobe Photoshop CS6 (Adobe Systems, New York, USA).