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The Sussex Centre for Advanced Microscopy

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Compiled by: Julian Thorpe

 

Immunogold Labelling (Publications)

For a diagrammatic representation of the process of immungold labelling, please start here

(Right)  An example of the immunogold localisation of a neuropeptide in the snail  heart (note the accumulations of gold  particles over the neural granules).

In the EM laboratory here there is considerable experience and expertise in the area of immunogold labelling. Many research projects within the School have been aided by the precise localisation at the ultrastructural level of particular proteins under study (see refs. below).
To summarise the technique briefly, specimens are prepared in a manner such that the antigenicity of their proteins is maintained (for more detail click here).
Thin sections of these samples are then incubated with an (primary) antibody raised to the specific antigen and the sites of antibody-binding are made visible in the TEM by incubation in a secondary antibody (with an affinity for the primary antibody) to which ultra-small (typically 5 or 10nm) gold particles have been attached (click here for further experimental detail).
Subsequent images taken on the TEM reveal the localisation of the gold particles and thus the antigen under investigation (for examples, see image gallery).

Some Reference Examples from the School of Life Sciences:

Soura V, Stewart Parker M, Williams TL, Ratnayaka A, Atherton J, Gorringe K, Tuffin J, Darwent E, RambaranR, Klein W, Lacor P, Staras K, Thorpe J, Serpell L (2012) Visualisation of colocalisation in Ab42-administered neuroblastoma cells reveals lysosome damage and autophagosome accumulation related to cell death. Biochemical Journal  441:579-590

Page T, Gitcho MA, Mosaheb S, Carter D,Chakraverty S, Perry RH, Bigio EH, Gearing M, Ferrer I, Goate AM, Cairns NJ, Thorpe JR (2011) FUS immunogold labelling TEM analysis of the neuronal cytoplasmic inclusions of neuronal intermediate filament inclusion disease: a frontotemporal lobar degeneration with FUS proteinopathy. Journal of Molecular Neuroscience 45: 409-421 DOI: 10.1007/s12031-011-9549-8

Acevedo-Arozena A, Kalmar B, Essa S, Ricketts T, Joyce P, Kent R, Rowe C, Parker A, Gray A, Hafezparast M, Thorpe JR, Greensmith L, Fisher EMC (2011)  A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis. Disease Models & Mechanisms 4: 686-700  

Caberlotto E, Michel V, Foucher I, Bahloul A, Goodyear RJ, Pepermans E, Michalski N, Perfettini I, Alegria-Prevot O, Chardenoux S, Do Cruzeiro M, Hardelin JP, Richardson GP, Avan P, Weil D, Petit C (2011) Usher type 1G protein sans is a critical component of the tip-link complex, a structure controlling actin polymerization in stereocilia. Proceedings of the National Academy of Sciences of the United States of America 108: 5825-5830

Nayak G, Goodyear RJ, Legan PK, Noda M, Richardson GP (2011) Evidence for multiple, developmentally regulated isoforms of Ptprq on hair cells of the inner ear. Developmental Neurobiology 71: 129-141

Verpy E, Leibovici M, Michalski N, Goodyear RJ, Houdon C, Weil D, Richardson GP, Petiti C (2011) Stereocilin connects outer hair cell stereocilia to one another and to the tectorial membrane. Journal of Comparative Neurology 519: 194-210

Richardson GP,de Monvel JB, Petit C (2011) How the genetics of deafness illuminates auditory physiology. Annual Reviews of Physiology 73: 311-334

Acevedo-Arozena A, Kalmar B, Essa S, Ricketts T, Joyce P, Kent R, Rowe C, Parker A, Gray A, Hafezparast M, Thorpe JR, Greensmith L, Fisher EMC (2011)  A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis. Disease Models & Mechanisms 4 (In Press)  

Goodyear RJ, Forge A, Legan PK, Richardson GP (2010) Asymmetric distribution of cadherin 23 and protocadherin 15 in the kinocilial links of avian sensory hair cells. Journal of Comparative Neurology 518: 4288-4297

Paine S, Bedford L, Thorpe JR, Mayer RJ, Bajaj N, Sheppard PW, Lowe J, Layfield R (2009) Immunoreactivity to Lys63-linked polyubiquitin is a feature of neurodegeneration. Neuroscience Letters 460: 205-208

Goodyear RJ, Gale JE, Ranatunga KM, Kros CJ, Richardson GP. (2008) Aminoglycoside-induced phosphatidylserine externalization in sensory hair cells is regionally restricted, rapid, and reversible. J Neurosci. 28(40):9939-52 PMID: 18829952 [PubMed - indexed for MEDLINE]

van Aken AF, Atiba-Davies M, Marcotti W, Goodyear RJ, Bryant JE, Richardson GP, Noben-Trauth K, Kros CJ. (2008) TRPML3 mutations cause impaired mechano-electrical transduction and depolarization by an inward-rectifier cation current in auditory hair cells of varitint-waddler mice. J Physiol. 586(Pt 22):5403-18 PMID: 18801844 [PubMed - indexed for MEDLINE

Thorpe JR, Tang HHL, Atherton JM, Cairns NJ (2008) Fine structural analysis of the neuronal inclusions of frontotemporal lobar degeneration with TDP-43 proteinopathy. Journal of Neural Transmission 115: 1661-1671

Hashemzadeh-Bonehi L, Phillips RG, Cairns NJ, Mosaheb S, Thorpe JR (2006) Pin1 protein associates with neuronal lipofuscin: potential consequences in age-related neurodegeneration. Experimental Neurology  199: 328-338 

McGee J, Goodyear RJ, McMillan DR, Stauffer EA, Holt JR, Locke KG, Birch DG, Legan PK, White PC, Walsh EJ, Richardson GP (2006) The Very Large G-Protein-Coupled Receptor VLGR1: A Component of the Ankle Link Complex Required for the Normal Development of Auditory Hair Bundles. Journal of Neuroscience 26: 6543-6553

Ahmed ZM, Goodyear R, Riazuddin S, Lagziel A, Legan PK, Behra M, Burgess SM, Lilley KS, Wilcox ER, Riazuddin S, Griffith AJ, Frolenkov GI, Belyantseva IA, Richardson GP, Friedman TB (2006) The Tip-Link Antigen, a Protein Associated with the Transduction Complex of Sensory Hair Cells, Is Protocadherin-15. Journal of Neuroscience 26: 7022-7034  

Mosaheb S, Thorpe JR, Hashemzadeh-Bonehi L, Bigio EH, Gearing M, Cairns NJ (2005) Neuronal intranuclear inclusions are ultrastructurally and immunologically distinct from cytoplasmic inclusions of neuronal intermediate filament inclusion disease (NIFID). Acta Neuropathologica 110: 360-368 

Thorpe JR, Mosaheb S, Hashemzadeh-Bonehi L, Cairns NJ, Kay KE, Morley SJ, Rulten S (2004) Shortfalls in the Peptidyl-Prolyl Cis-Trans Isomerase Protein Pin1 in Neurons are Associated With Frontotemporal Dementias. Neurobiology of Disease 17: 237-249

Cairns NJ, Grossman M, Arnold SE, Burn DJ, Jaros E, Perry RH, Duyckaerts C, Stankoff B, Pillon B, Skullerud K, Cruz-Sanchez FF, Bigio EH, Mackenzie IRA, Gearing M, Juncos JL, Glass JD, Yokoo H, Nakazato Y, Mosaheb S, Thorpe JR, Uryu K, Lee V.M.-Y, Trojanowski, JQ (2004) Clinical and neuropathologic variation in neuronal intermediate filament inclusion disease (NIFID). Neurology 63: 1376-1384                    

Thorpe JR, Morley SJ, Rulten SL (2001) Utilising the Peptidyl-Prolyl Cis-Trans Isomerase Pin1 as a Probe of its Phosphorylated Target Proteins: Examples of Binding to Nuclear Proteins in a Human Kidney Cell Line and to Tau in Alzheimer’s Diseased Brain. J. Histochem. Cytochem. 49: 97-108  

Thorpe JR, Rulten SL, Kay JE (1999) The binding of a putative and a known chaperone protein revealed by immunogold labelling transmission electron microscopy: A suggested use of chaperones as probes for the distribution of their target proteins. J. Histochem. Cytochem. 47, 1633-1640  

Rulten SL, Thorpe JR, Kay JE (1999) Identification of Eukaryotic Parvulin Homologues: A new subfamily of peptidyl-prolyl cis-trans isomerases. Biochem  Biophys Res Comm 259: 557-562  

Thorpe, J.R. (1999) The Application of LR Gold Resin for Immunogold Labelling. Chapter 6 in Electron Microscopy Methods and Protocols. Ed. M.A. Hajibagheri. Methods in Molecular Biology Series. Humana Press Inc. USA Vol. 117 pp. 99-110

Thorpe, J.R. (1992) A novel methodology for double protein A-gold immunolabelling utilising the monovalent fragment of protein A. Journal of Histochemistry and Cytochemistry 40, 435-441

Moore, A.L., Walters, A.J., Thorpe, J.R. Fricaud, A-C. and Watts, F.Z. (1992) Schizosaccharomyces pombe mitochondria: morphological, respiratory and protein import characteristics. Yeast 8, 923-933

Thorpe, J.R. and Wallis, M. (1991) Immunocytochemical and morphometric investigations of mammotrophs, somatotrophs and somatomammotrophs in sheep pituitary cell cultures. Journal of Endocrinology 129: 417-422

Thorpe, J.R., Ray, K.P. and Wallis, M. (1990) Occurrence of rare somatomammotrophs in ovine anterior pituitary tissue studied by immunogold labelling and electron microscopy. Journal of Endocrinology 124: 67-73

Khawaja, X.Z., Green, I.C., Thorpe, J.R. and Bailey, C.J. (1990) Increased sensitivity to insulin-releasing and glucoregulatory  effects of  dynorphin-A1-13 and U-50488H in OB/OB versus lean mice. Diabetes 39: 1289-1297

Khawaja, X.Z., Green, I.C., Thorpe, J.R. and Titheradge, M.A. (1990) The occurrence and receptor specificity of endogenous opioid peptides within the pancreas and liver of the rat - comparison with brain. Biochemical Journal 267: 233-240

Tobin, A.K., Thorpe, J.R., Hylton, C.M. and Rawsthorne, S. (1989) Spatial and temporal influences on the cell-specific localisation of glycine decarboxylase in leaves of wheat (Triticum aestivum) and pea (Pisum sativum). Plant Physiology 91, 1219-1225