Ecl prime detection agent

The cultured cells were xed by treatment with 4% formaldehyde for 5 minutes. The membrane at the bottom of the insert cup was cut with a scalpel, transferred to a slide glass, and encapsulated with mounting medium (50% glycerol, 0.05% NaN 3 in phosphate buffered saline). Then, the specimen was observed with a uorescence microscope (Olympus BX53-FK, exposure time: 10 milliseconds), and the ratio of BODIPY TM FL C 16 that accumulated in areas containing lipid droplets was analyzed with Olympus cellSens.
Evaluation of secreted ApoB-48 and ApoB-100 by western blotting Basal side culture medium was collected and mixed with ×1.25 sample buffer (2.08% sodium lauryl sulfate, 6.25% glycerol, 1.94% dithiothreitol in 0.073 M Tris-HCl buffer, pH 6.8) and denatured by boiling for 5 minutes. The protein was subjected to 5% SDS-PAGE and transferred to a PVDF membrane. The membrane was treated with blocking agent, and labeled with a mouse anti-human ApoB monoclonal antibody (7B8) (ab39560, Abcam plc, Cambridge, UK) as the primary antibody, and horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody (Nr.P.0447, Dako, Agilent Technologies, Santa Clara, CA) as the secondary antibody, which was detected by ECL prime detection agent (GE Healthcare, Little Chalfont, UK). The resulting images were analyzed with Image J (version 1.52a).