Sections were incubated in serum free block (DAKO UK Ltd, Ely, Cambridgeshire, UK) to block nonspecific background staining then endogenous biotin was blocked with an Avidin/Biotin blocking agent (Vector Laboratories, Peterborough, UK). however, demonstrated generally higher levels of cytoplasmic staining (concordance 74.77%, kappa 0.351). The antibodies demonstrated very different patterns of nuclear staining. Over 60% of patients stained with the H4.77.16 had no Povidone iodine nuclear staining whereas the vast majority showed staining with the HFR1 antibody (concordance 40.12%, kappa 0.051). Neither antibody demonstrated relationships between membranous or cytoplasmic HER4 staining and survival, although associations EDC3 were seen with known poor prognostic markers. Cases with H4.77.16-determined nuclear staining had significantly poorer survival outcomes. Conclusion The difference in antigen site may explain the different staining patterns we have seen with respect to location; with each antibody appearing to select for distinct compartments. Thus, HFR1 may select for cytoplasmic and nuclear HER4 ICD, Povidone iodine whilst H4.77.16 selects for membranous HER4 and/or HER4 being recycled in cytoplasm or nucleus. This ability to distinguish between site and function of HER4 and its fragments is particularly important, with recent evidence highlighting the different functions Povidone iodine of nuclear and mitochondrial HER4. Introduction Overexpression of members of the human epidermal growth factor receptor (HER) family has been widely studied in breast cancer. Whereas the biology underlying the role of HER2 and epidermal growth factor receptor (EGFR) has been increasingly documented, more confusion exists in establishing a role for HER4 (c-erbB-4). We have shown that, in contrast to other HER family members, HER4 expression is associated with increased survival and lower proliferation indices [1,2]. These results are Povidone iodine supported by data linking HER4 to established good prognostic indicators such as a lower grade of tumour [3,4], oestrogen receptor (ER) positivity [5] and low proliferation indices [6]. However, whilst other groups have also demonstrated a link between HER4 positivity and a longer disease free interval [7], conflicting reports have associated HER4 Povidone iodine with an adverse prognostic significance [8]. More recently, evidence from a large series of patients has suggested that the prognostic value of HER4 overexpression is dependent on coexpression with other HER family members [9]. In this study, when the group was looked at as a whole, HER4 status was not related to survival [9]. In cases showing expression of one family member only (homodimers), however, they found a significant association between HER4 homodimer-expressing tumours and improved disease free survival. There are intrinsic problems in comparing these studies and their outcomes. Different cut off points for positivity have been chosen depending on the study and the modality of staining looked at (membrane, cytoplasm and nuclear). Some groups have reported staining in all three locations, whilst others have found no membranous [8] or no nuclear staining [7]. Three different antibodies have been used in these studies. The HFR1 clone developed by the Gullick group has been the most widely used [3,4,8-10]. This group demonstrated the ability of this antibody to recognise HER4 by immunoprecipitation, western blotting and immuno-staining of NH3T3 cells transfected with HER4. They demonstrated no cross-reactivity with EGFR using A431 cell lysates or with HER3 or HER4 using lysates from SKBR3 or 293/HER3 cells. A Santa Cruz antibody C18 has also been used by one group [7]. In our previously study on frozen tissue, we used a Neomarkers antibody H4.77.16 [1]. Recent studies have substantially enhanced our understanding of the many functions of HER4. Indeed, as well as acting as a membrane signal transduction receptor, nuclear HER4 is required for mammary gland development and lactation through gene regulation in conjunction with STAT5A [11,12], and mitochondrial HER4 has been shown to mediate apoptosis in.