D (I). Bars represent 200 m. Semiquantitative analysis of staining intensity for FLOT1 in GE (J). Data represented as mean ?SEM. * P 0.05, ** P 0.01, *** P 0.001, Kruskal Wallis test, with Dunn's post-hoc test.Yap et al. Reproductive Biology and Endocrinology 2011, 9:73 http://www.rbej.com/content/9/1/Page 13 ofImmunostaining- Flotillin-1 (FLOT1)Primary hEEC cellsABCEEC-1 cellsDEFFigure 7 Photomicrograph representing immunocytochemical staining for flotillin-1 (FLOT1) in human endometrial epithelial cells (hEEC). Primary hEEC and the cell lines ECC-1 were treated with diluent or IL11 (100 ng/ml) for 24 h. Cytospins were prepared and subjected to immunocytochemistry as described in the Materials Methods. Positive staining for FLOT1 showed as brown pigment with blue nuclear counterstain. (A, D) Negative IgG (B) Control primary EEC (C) IL11 100 ng/ml primary hEEC (E) Control ECC-1 (F) IL11 100 ng/ml ECC-1. Bars represent 200 m.this is due to the high levels of FLOT1 found in the epithelium during the secretory stage, thus, it was not possible to detect an increase in FLOT1 expression, whereas, the absence of FLOT1 in the proliferative stage epithelium allowed detection of FLOT1 stimulated following IL11 treatment. Further, we suggest we were unable to detect FLOT1 in primary hEEC by Western blot because of the unfeasibly large number of cells required to detect a low abundance protein (as demonstrated by cytospin immunolocalisation) such as this. FLOT1 is a lipid raft associated membrane protein and is endocytosed from the plasma membrane into intracellular compartments [28]. FLOT1 has also been shown to reside in the plasma membrane, as well as in the cytoplasm [29] as observed in our cytospin data.Recent findings demonstrate that FLOT1 is associated with cell PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6811518 motility and transformation, membrane trafficking, phagocytosis and epidermal growth factor receptor signaling [30]. Given that FLOT1 localises to the cell membrane lipid rafts, it is not surprising that it is also involved in cellular communication, such as cell-cell contacts, focal adhesions, the T-cell cap, and synapses [31]. Previous studies in several cell lines indicate that FLOT1 localises to plasma membranes [32-35]. In our study, FLOT1 immunostaining was primarily punctate within the plasma membranes of hEEC and ECC-1 cells and also in the glandular and luminal epithelial cells in human endometrium. This is in agreement with a previous study that identified FLOT1 in punctate structures or microdomains in plasma membranes of COS-7 cells,Yap et al. Reproductive Biology and Endocrinology 2011, 9:73 http://www.rbej.com/content/9/1/Page 14 ofsuggesting the recruitment/localisation of FLOT1 to the membrane compartments [36]. The FLOT1 punctate staining was primarily present apically in glandular and luminal epithelial cells from mid-secretory or receptive phase endometrium. In late-secretory phase endometrium, FLOT1 staining was present basolaterally in the glands and did not appear punctate. This may have been due to the overall lower level of staining seen in late secretory phase compared to mid-secretory phase endometrium, although it did not reach statistical significance. Studies carried out by our Capecitabine group in the past have showed that IL11 localises in the glandular epithelium and that it may be secreted apically into uterine lumen and basally into the endometrium [10]. It is tempting to speculate that the presence of FLOT1 in combination with IL11 facilitates cell adhesi.