Three-dimensional immunofluorescent double labelling using polyclonal antibodies derived from the same species: enterocytic colocalization of chylomicrons with Golgi apparatus

Abstract

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.

Aging-Related Changes in Blood-Brain Barrier Integrity and the Effect of Dietary Fat

“…In this study, we used an established, highly sensitive immunofluorescent microscopy procedure to quantitatively assess the parenchymal abundance of IgG [15,24]. In addition, colocalization analysis with a specific marker of cerebrovascular inflammation (GFAP) was carried out in 3 dimensions.…”

“…This is a widely used and established method [22,23], and in this study we used published quantitative 3-dimensional (3-D) immunomicroscopy methods described previously [8,9,15,24,25,26], with minor modifications. The right hemisphere of the brain was carefully isolated and washed in ice-cold PBS.…”

“…The abundance of the cerebrovascular tight junction protein occludin-1 relative to the endothelium was quantitatively determined in the cortex and hippocampus using 3-D double immunofluorescent microscopy as described previously [15,24]. Briefly, the brain cryosections (20 µm thick) were fixed with acetone at -20°C for 3 min, and heat-mediated antigen retrieval was conducted by incubating the sections in 60°C water for 2 h. Endogenous biotin was blocked with avidin in egg white and biotin in skimmed milk.…”

State-of-the-art technologies, current opinions and developments, and novel findings: news from the field of histochemistry and cell biology

“…Subjects covered in recent original papers and reviews in this Weld were the generation and application of speciWc antibodies suited to detect speciWc antigens and/or functional states of macromolecules, glycolipids, advanced glycation end products, and small molecules (e.g., Diamanti-Kandarakis et al 2007;Fujiwara et al 2007;Ohara et al 2007a, b;Schmidt et al 2007;Young et al 2007;Kolling et al 2008;Mandell 2008;Myöhänen et al 2008), the optimization of detection protocols including tissue preparation, improvement of antigen accessibility, and modiWcations of immunocytochemical procedures (Schlormann et al 2007;Blaschitz et al 2008;Takechi et al 2008), the improvement of methods for qualitative and quantitative analyses of the subcellular localization of proteins using immunogold labelling (Bowes et al 2007;Lopez et al 2008;Mayhew and Lucocq 2008a, b), and the quality control in molecular immunohistochemistry (True 2008). In particular, a problem with which the scientist is often confronted in double immunolabelling was addressed in an investigation by Takechi et al (2008): often, the best antibodies available against antigens of interest have been generated in the same species, and thus double labelling appears not applicable at least in indirect immunoXuorescence since cross-reactions of the secondary antibodies will necessarily occur. In their study, the authors developed a protocol for double immunolabelling using rabbit-derived polyclonal antibodies by applying a high dilution for the Wrst antibody so that detectability required biotin-avidin ampliWcation, and by visualizing the second antibody via a standard secondary antibody protocol which was not suYciently sensitive to detect the Wrst antibody.…”

Probucol Suppresses Enterocytic Accumulation of Amyloid‐β Induced by Saturated Fat and Cholesterol Feeding

“…Intestinal Aβ and apo B were detected by immunoflourescent amplification method as previously described [31]. Intestinal tissue sections (5 μm) were deparaffinised, rehydrated and antigen-retrieval was carried out in boiling deionised water for 15 min.…”