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Danbolt, NC, Zhou, Y, Furness, DN and Holmseth, S (2016) Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons. Glia, 64 (12). pp. 2045-2064.

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Abstract

Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control.

Item Type:	Article
Additional Information:	This is the peer reviewed version of the following article: Strategies for immunohistochemical protein localization using antibodies: what did we learn from neurotansmitter transporters in glial cells and neurons, which has been published in final form at 10.1002/glia.23027. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving
Uncontrolled Keywords:	antibody specificity; immunocytochemistry; western blotting; glutamate uptake; excitatory amino acid transporter
Subjects:	R Medicine > R Medicine (General)
Divisions:	Faculty of Natural Sciences > School of Life Sciences
Depositing User:	Symplectic
Date Deposited:	01 Aug 2016 09:14
Last Modified:	10 Apr 2019 08:41
URI:	http://eprints.keele.ac.uk/id/eprint/2079

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