Vimentin clone v9

Confluent adipocyte monolayers were washed with PBS and stained with AdipoRed (Lonza) in PBS (1:33) for 15 min at room temperature (RT) according to the manufacturer’s instructions before imaging. Tissue samples were embedded in paraffin and 5 µm sections were used for morphological (haematoxylin & eosin, H&E) and immunohistochemical stainings. Antibodies for cytokeratin 15 (K15, clone EPR1614Y; Abcam, Cambridge, UK), cytokeratin 10 (K10, clone DE-K10; Progen, Heidelberg, Germany) and vimentin (clone V9; Dako, Glostrup, Denmark) were used as previously described [27 (link), 28 (link)]. Prior to embedding in Aquatex (Merck), sections were counter-stained with hematoxylin. For confocal imaging, tissue samples were fixed in 4% formaldehyde overnight, stored in PBS with 0.02% sodium azide (Merck) and subsequently stained with AdipoRed (1:33) and DAPI (1:5000) for 15 min at RT.

Immunohistochemical labeling was performed according to standard protocols on serial 2.5 μm thick sections from the original blocks or the TMA blocks. All cases were also stained for vimentin (clone V9, Dako, Glostrup, Denmark) and cytokeratin 8/18 (clone 5D3, Novocastra^TM, Leica Biosystems, Newcastle, U.K), which were used as control stains for tissue immunoreactivity and fixation, as well as identification of tumor cells. Tissue samples negative for the above antibodies (21 of 975, 2.2%) were excluded from the study. To assure optimal reactivity, immunostaining was applied 7 to 10 days after sectioning at the Laboratory of Molecular Oncology of the Hellenic Foundation for Cancer Research, Aristotle University of Thessaloniki School of Medicine. The staining procedures for vimentin, cytokeratin 8/18, HER2 (A0485 polyclonal antibody, Dako), estrogen receptor (ER, clone 6F11, Novocastra^TM, Leica Biosystems), progesterone receptor (PgR, clone 1A6, Novocastra^TM, Leica Biosystems) and Ki67 (clone MIB-1, Dako) were performed using a Bond Max^TM autostainer (Leica Microsystems, Wetzlar, Germany), as previously described in detail [33 (link)–35 (link)].

Four renal tumours from Fischer male rats given protracted dietary OTA (300 µg/kg body weight daily) [6 (link)] were embedded in paraffin blocks. Animals were from the same lifetime experimental group [6 (link)] as those whose immunoprofiles were previously described [9 (link)]. Ethical review and approval were waived for this study because no new live animals were involved. Sections (3 μm) were mounted on charged slides (TOMO, Matsunami, Japan) and processed for immunohistochemistry in the Cell Pathology Laboratory of South West London Pathology at St George’s Hospital, Tooting, variously applying panels of antibodies in fully automated BenchMark ULTRA immunohistochemistry processing, as required to assist clinical diagnoses. Procedures followed exactly those previously described [9 (link)]. The following antibodies were used: CK MNF 116, clone MNF 116 (Dako); Vimentin, clone V9 (Dako, Novocastra); CD10, clone 56C6 (Dako). After applying DAB chromogen, nuclei were counterstained blue with haematoxylin. The brown immune reaction product of DAB chromogen is cytoplasmic and/or membranar for the listed antibodies. Haematoxylin and Eosin staining was also performed for preliminary standard tissue differentiation of nuclear (blue) and cytoplasmic components (red).