Three-dimensional immunofluorescent double labelling using polyclonal antibodies derived from the same species: enterocytic colocalization of chylomicrons with Golgi apparatus
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Double immunolabelling is a useful technique to determine cellular colocalization of proteins, but is prone to false-positive staining because of cross-reactivity between antibodies. In this study, we established a simple and quick method to demonstrate the immunofluorescent double labelling with two rabbit-derived polyclonal antibodies. The principle used was to establish a dilution of primary antibody for the first protein of interest, which would only be detectable following biotin–avidin amplification. Thereafter, the second protein of interest was assessed via standard secondary antibody detection, ensuring no cross-reactivity with the first protein antibody–antigen complex. We successfully demonstrated the three-dimensional colocalization of enterocytic apolipoprotein B, an equivocal marker of intestinal lipoproteins with Golgi apparatus. Colocalization of apo B and Golgi apparatus (75.2 ± 8.5%) is consistent with the purported mode of secretion of these macromolecules.
KeywordsDouble immunofluorolabelling Three-dimensional colocalization Polyclonal antibodies Intestinal Golgi apparatus Chylomicron
This project was kindly supported by the Australian Technology Network, Centre for Metabolic Fitness. R. Takechi is supported by a Curtin University of Technology, International Research Tuition Scholarship. Mr. Mark Potten (Zeiss, Australia) is acknowledged for support of the Faculty of Health Sciences microscopy facilities at Curtin University.
Fig. S1 Movie file of 3D localization. Three-dimensional colocalization of apo B (yellow) and Golgi apparatus (red) was also captured as movie file by using AxioVision (MOV 7155 kb)
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