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Multiple-labelling immunoEM using different sizes of colloidal gold: alternative approaches to test for differential distribution and colocalization in subcellular structures

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Abstract

Various methods for quantifying cellular immunogold labelling on transmission electron microscope thin sections are currently available. All rely on sound random sampling principles and are applicable to single immunolabelling across compartments within a given cell type or between different experimental groups of cells. Although methods are also available to test for colocalization in double/triple immunogold labelling studies, so far, these have relied on making multiple measurements of gold particle densities in defined areas or of inter-particle nearest neighbour distances. Here, we present alternative two-step approaches to codistribution and colocalization assessment that merely require raw counts of gold particles in distinct cellular compartments. For assessing codistribution over aggregate compartments, initial statistical evaluation involves combining contingency table and chi-squared analyses to provide predicted gold particle distributions. The observed and predicted distributions allow testing of the appropriate null hypothesis, namely, that there is no difference in the distribution patterns of proteins labelled by different sizes of gold particle. In short, the null hypothesis is that of colocalization. The approach for assessing colabelling recognises that, on thin sections, a compartment is made up of a set of sectional images (profiles) of cognate structures. The approach involves identifying two groups of compartmental profiles that are unlabelled and labelled for one gold marker size. The proportions in each group that are also labelled for the second gold marker size are then compared. Statistical analysis now uses a 2 × 2 contingency table combined with the Fisher exact probability test. Having identified double labelling, the profiles can be analysed further in order to identify characteristic features that might account for the double labelling. In each case, the approach is illustrated using synthetic and/or experimental datasets and can be refined to correct observed labelling patterns to specific labelling patterns. These simple and efficient approaches should be of more immediate utility to those interested in codistribution and colocalization in multiple immunogold labelling investigations.

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Acknowledgments

We are grateful to fellow teachers on the EMBO-sponsored Practical Courses in Electron Microscopy and Stereology in Cell Biology. They have been a constant source of stimulation and encouragement. Our thanks also go to Professor Stephen McKenna (School of Computing, University of Dundee) for helpful discussion on alternative statistical models.

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Authors and Affiliations

  1. School of Biomedical Sciences, Queen’s Medical Centre, University of Nottingham, E Floor, Nottingham, NG7 2UH, UK

    Terry M. Mayhew

  2. Division of Cell Biology and Immunology, School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK

    John M. Lucocq

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  1. Terry M. Mayhew
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Correspondence to Terry M. Mayhew.

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Mayhew, T.M., Lucocq, J.M. Multiple-labelling immunoEM using different sizes of colloidal gold: alternative approaches to test for differential distribution and colocalization in subcellular structures. Histochem Cell Biol 135, 317–326 (2011). https://doi.org/10.1007/s00418-011-0788-0

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