Goals and practicalities of immunoblotting and immunohistochemistry: A guide for submission to the British Journal of Pharmacology

Abstract

Reproducibility is a current concern for everyone involved in the conduct and publication of biomedical research. Recent attempts testing reproducibility, particularly the reproducibility project in cancer biology published in elife (https://elifesciences.org/collections/9b1e83d1/reproducibility-project-cancer-biology), have exposed major difficulties in repeating published preclinical experimental work. It is thought that some of these difficulties relate to uncertainty about the provenance of tools, lack of clarity in methodology and use of inappropriate approaches for analysis; the latter particularly related to untoward manipulation of images. In the past, some of these so-called untoward practices were considered the 'norm'; however, today, the landscape is different. The expectations, not only of the readers of the published scientific word but also of the publishers and funders of research, have changed. This collective group now expects that any published data should be reproducible; but for this to be possible, experimental detail, confirmation of selectivity and quality of reagents/ tools, analytical and statistical methods used need to be described adequately. Two powerful methodologies often used to support researchers' findings allow the detection of changes in protein expression, that is, immunoblotting (widely known as Western blotting) and immunohistochemistry. Undeniably, as a result of unintentional mistakes (often related to lack of antibody specificity; Baker, 2015), but, in some cases, deliberate alterations and questionable interpretations of results, the use of these two methods has led to many high profile retractions. Indeed, such images have driven the retractions that have occurred in BJP over the last two years.Today, immunoblotting and immunohistochemistry serve as primary methodologies for the detection and quantification of molecular signalling pathways and identification of therapeutic targets. This necessitates clear guidance for the application of these techniques, the need for controls (both positive and negative) and the most appropriate methods for quantification. Indeed, this need has spawned a number of initiatives to support researchers in assessing the validity of antibody resources including antibodypedia (Bjorling and Uhlen, 2008) and the resources available within 'The Human Protein Atlas' (Thul et al., 2017). The aim of this article is to outline the rationale for, and the expectations of, the BJP with respect to work published in the Journal that includes immunoblotting or immunohistochemical data. In creating these guidelines, our aim is to reduce potential misinterpretations and to maximise the communication and transparency of essential information, particularly with respect to the methodologies employed.We have generated the guidelines below for the benefit of authors, editors and reviewers. While we recognise other recently published guidelines (Uhlen et al., 2016) and indeed we have incorporated some of the advice provided in such reports, we focus, here, on th...

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