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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.

Original publication

DOI

10.1007/s00418-008-0404-0

Type

Journal article

Journal

Histochem Cell Biol

Publication Date

06/2008

Volume

129

Pages

779 - 784

Keywords

Animals, Antibodies, Apolipoproteins B, Chylomicrons, Enterocytes, Female, Golgi Apparatus, Immunohistochemistry, Intestines, Mice, Mice, Inbred C57BL