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 the evidence required for publication in BJP. These guidelines join a series published in BJP regarding the reporting of animal experiments through adoption of the ARRIVE guidelines (McGrath et al., 2015; McGrath and Lilley, 2015), experimental design and analysis (Curtis et al., 2015) and data sharing and presentation (George et al., 2017) in preclinical pharmacology. We would be delighted if other journals were to also use these guidelines. If an antibody has been characterised previously, a citation must be included. This characterization should fulfil the requirements detailed in the recent guidelines generated by ‘The International Working Group for Antibody Validation’ and should take the form of at least, but preferably more than, one of the ‘five conceptual pillars of antibody validation’ described in the guidelines (Uhlen et al., 2016). We also ask authors to check currently available databases for known issues with selectivity. Currently, we highly recommend the NIH-established Research Resource Identification Protocol (RRID) at https://scicrunch.org/resources/Cell%20Lines/search, which provides a unique identifier for the antibody that enables reproducibility studies through clear indication of provenance. Although encouraged, the inclusion of an RRID is not currently mandatory for BJP; we envisage that a similar level of individualization will become essential for publication in the near future. To support the validity of conclusions emanating from immunoblotting or immunohistochemical observations (as with the majority of biological assays), it is important to conduct both positive and negative controls – although for some proteins, certain positive and negative controls are unknown or such materials are not available, thereby precluding such assessments. However, there are a number of other ‘method’ controls that are always possible and which should be included in assay protocols. We recognise that there may be no perfect control that provides confidence beyond doubt (Torlakovic et al., 2015). In this digital age, a growing concern amongst the scientific community is that images for publication are not being accurately presented, that is, the problem of unintentional and/or inappropriate manipulation of images (Cromey, 2010). For example, while it is usually acceptable to crop an image to simplify the information, choosing to crop out oversaturated regions and/or areas/bands displaying non-specific immunoreactivity is not acceptable. BJP requires submission of full immunoblot scans and immunohistochemical/fluorescent images, from which figures have been generated. These scans/images should be included as an additional file for the review process and will be used by the Editors and reviewers when assessing the manuscript, but need not be published. In instances where uncertainty occurs regarding image compilation and assembly during the review process, the BJP Editorial Office makes use of freely available Office of Research Integrity Forensic tools (https://ori.hhs.gov/forensic-tools), developed by the US Department of Health and Human Services. Conforming to these stipulations will ensure that reviewers and readers can confirm that the band identified is at the correct molecular size (or allow some interpretation of post-translational modifications, for example) and to determine the selectivity of the antibody used. For any antibody (including secondary antibodies) used, the Methods section should include the following: