Three-dimensional immunofluorescent double labelling using polyclonal antibodies derived from the same species: enterocytic colocalization of chylomicrons with Golgi apparatus.
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.
Takechi
R
R
Australian Technology Centre for Metabolic Fitness, School of Public Health, Division of Health Sciences, Curtin University of Technology, GPO Box U1987, Perth, WA, 6845, Australia.
Galloway
S
S
Pallebage-Gamarallage
M M S
MM
Johnsen
R D
RD
Mamo
J C L
JC
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001055:Apolipoproteins B / Q000276:immunology / Q000378:metabolism; D002914:Chylomicrons / Q000276:immunology* / Q000378:metabolism; D020895:Enterocytes / Q000276:immunology* / Q000648:ultrastructure; D005260:Female; D006056:Golgi Apparatus / Q000276:immunology* / Q000378:metabolism; D007150:Immunohistochemistry; D007422:Intestines / Q000276:immunology / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL
D000906:Antibodies; D001055:Apolipoproteins B; D002914:Chylomicrons