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

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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.

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References

  • Bauer M, Schilling N, Spanel-Borowski K (2001) Limitation of microwave treatment for double immunolabelling with antibodies of the same species and isotype. Histochem Cell Biol 116:227–232

    PubMed  CAS  Google Scholar 

  • Galloway S, Jian L, Johnsen R, Chew S, Mamo JC (2007) β-amyloid or its precursor protein is found in epithelial cells of the small intestine and is stimulated by high-fat feeding. J Nutr Biochem 18:279–284

    Article  PubMed  CAS  Google Scholar 

  • Hermiston ML, Latham CB, Gordon JI, Roth KA (1992) Simultaneous localization of six antigens in single sections of transgenic mouse intestine using a combination of light and fluorescence microscopy. J Histochem Cytochem 40:1283–1290

    PubMed  CAS  Google Scholar 

  • Hunyady B, Krempels K, Harta G, Mezey E (1996) Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining. J Histochem Cytochem 12:1353–1362

    Google Scholar 

  • Hussain MM (2000) A proposed model for the assembly of chylomicrons. Atherosclerosis 148:1–15

    Article  PubMed  CAS  Google Scholar 

  • Kroeber S, Schomerus C, Korf H-W (1998) A specific double-immunofluorescence method for the demonstration of S-antigen and serotonin in trout and rat pinealocytes by means of primary antibodies from the same donor species. Histochem Cell Biol 109:309–317

    Article  PubMed  CAS  Google Scholar 

  • Nishimura Y, Yoshioka K, Bernard O, Bereczky B, Itoh K (2006) A role of LIM kinase 1/cofilin pathway in regulating endocytic trafficking of EGF receptor in human breast cancer cells. Histochem Cell Biol 126:627–638

    Article  PubMed  CAS  Google Scholar 

  • Rambozzi L, Menzano A, Lavin S, Rossi L (2004) Biotin-avidin amplified ELISA for detection of antibodies to Sarcoptes scabiei in chamois (Rupicapra spp.). Vet Res 35:701–708

    Article  PubMed  CAS  Google Scholar 

  • Shindler KS, Roth KA (1996) Double immunofluorescent staining using two unconjugated primary antisera raised in the same species. J Histochem Cytochem 44:1331–1335

    PubMed  CAS  Google Scholar 

  • Soldani C, Croce ACC, Bottone MG, Fraschini A, Biggiogera M, Bottiroli G, Pellicciari C (2007) Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage. Histochem Cell Biol 128:485–495

    Article  PubMed  CAS  Google Scholar 

  • Tornehave D, Hougaard DM, Larsson L-I (2000) Microwaving for double indirect immunofluorescence with primary antibodies from the same species and for staining of mouse tissues with mouse monoclonal antibodies. Histochem Cell Biol 113:19–23

    Article  PubMed  CAS  Google Scholar 

  • Tsurui H, Nishimura H, Hattori S, Hirose S, Okumura K, Shirai T (2000) Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition. J Histochem Cytochem 48:653–662

    PubMed  CAS  Google Scholar 

  • Uchihara T, Kondo H, Akiyama H, Ikeda K (1995) Single-laser three-color immunolabeling of a histological section by laser scanning microscopy: application to senile plaque-related structures in post-mortem human brain tissue. J Histochem Cytochem 43:103–106

    PubMed  CAS  Google Scholar 

  • Uchihara T, Nakamura A, Nakayama H, Arima K, Ishizuka N, Mori H, Mizushima S (2003) Triple immunofluorolabeling with two rabbit polyclonal antibodies and a mouse monoclonal antibody allowing three-dimensional analysis of cotton wool plaques in Alzheimer disease. J Histochem Cytochem 9:1201–1206

    Google Scholar 

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Acknowledgments

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.

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Authors and Affiliations

  1. 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

    R. Takechi, S. Galloway, M. M. S. Pallebage-Gamarallage & J. C. L. Mamo

  2. Australian Neuromuscular Research Institute, QEII Medical Centre, Verdun Street, Nedlands, Perth, WA, 6009, Australia

    R. D. Johnsen

  3. Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Verdun Street, Nedlands, Perth, WA, 6009, Australia

    R. D. Johnsen

Authors
  1. R. Takechi
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  2. S. Galloway
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  3. M. M. S. Pallebage-Gamarallage
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  4. R. D. Johnsen
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  5. J. C. L. Mamo
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Corresponding author

Correspondence to J. C. L. Mamo.

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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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Takechi, R., Galloway, S., Pallebage-Gamarallage, M.M.S. et al. Three-dimensional immunofluorescent double labelling using polyclonal antibodies derived from the same species: enterocytic colocalization of chylomicrons with Golgi apparatus. Histochem Cell Biol 129, 779–784 (2008). https://doi.org/10.1007/s00418-008-0404-0

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  • DOI: https://doi.org/10.1007/s00418-008-0404-0

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