weight, resp. acid and basic polypeptides. Squamous, stratified epithelium is usually characterized by the expression of CK 5, which is found mainly in the basal cell layers and Ck 5 is associated with the proliferative poten- tial of these cells [2,3]. The intermediary cell layers show an additional expression of Ck's 1 and 10, which are regarded as signs of cellular differentiation [2]. In con- trast, glandular epithelia reveal the expression of low- molecular weight cytokeratins Ck's 8/18 and 19 as typical features – expression pattern of Ck 8/18 is rather uncom- mon in mature squamous epithelium. Ck 19 expressed heterogeneously in the basal cell layers of stratified squa- mous epithelium [2,3]. Suprabasal expression of Ck 19 seems to be correlated with premalignant transformation in oral epithelium [4]. The expression of vimentin is mainly regarded as sign of a mesenchymal differentiation. However, vimentin-positively has repeatedly reported in various carcinomas and was interpreted as sign of an epi- thelial-mesenchymal transition, indicating an increased metastatic potential [5-9]. Recently, it could be shown that the induction of Ck 8/18 expression in non-malignant buccal mucosa cells resulted in a significant change of phenotypic characteristics after Ck 8/18 transfection [10]. These changes included an increased cellular motility, which might give first hints for an increased tumour aggressiveness and poor patient prognosis. However, these findings have not been trans- ferred on a clinical level, yet. Therefore, the significance of these findings in a clinical context, especially in regard to the prognostic importance is unclear. It was therefore the aim of this study to evaluate the expression of intermediary filaments in SCC's in relation to clinico-pathological features, especially for prognostic purposes by the use of tissue micro arrays and immuno- histochemistry. Methods Patients with histologically proven squamous cell carci- noma of the oral floor treated surgically were eligible for the study. All patients underwent surgical treatment including radical resection of the whole tumour with a free histopathological margin of at least 4 mm from the Antibody Company Ck 5/6 Ck 8/18 Ck 1 Ck 10 Ck 14 Ck 19 Vimentin DAKO, Glostrup, Denmark Dianova, Hamburg, Germany Novocastra, Newcastle, UK DAKO, Glostrup, Denmark Dianova Hamburg, Germany Quartett GmbH, Berlin, Germany Ventana Inc., Tucson, USA Table 1: Antibodies, company, clone, pre-treatment and dilution of used antibodies. BMC Cancer 2006, 6:10 http://www.biomedcentral.com/1471-2407/6/10 Page 2 of 8 (page number not for citation purposes) tumour borders. Selective neck dissection of Level I, II, III and V was performed in case of suspect results in preoper- ative tumourstaging by computertomography or sonogra- phy examination or in case of tumour size over 2 cm, bilateral selective neck dissection was performed when the tumour spread the midline (according to the recommen- dation of Robins et al. 2002 [11]). Radiotherapy was per- formed when lymph node metastases were detected histologically. All tumours were classified postsurgically according to the TNM system 6th eds. UICC 2002 [12]. Method Tumour specimens of 308 patients were investigated for the expression of Ck 5/6, Ck 8/18, Ck 1, CK 10, Ck 14, Ck 19 and vimentin (Table 1) by means of the tissue micro array (TMA) technique. For tissue micro array (TMA) construction, two punch biopsies with a diameter of 0.6 mm from each donor block were taken and transferred into the new acceptor block. TMA construction was performed by using a special tissue micro array instrument (Beecher Instruments, New Jersey, USA), according to standard protocols [13,14]. Immunohistochemistry was performed on 4-µm-thick TMA sections. After deparaffinization and rehydration, endogenous peroxidase activity was blocked for 30 min- utes in methanol containing 0.3% hydrogen peroxide. After antigen retrieval, a cooling-off period of 20 minutes preceded the incubation of the primary antibody. Thereaf- ter, the catalyzed signal amplification system (DAKO, Glostrup, Denmark) was used for Ck 5/6, Ck 8/18, Ck 1, CK 10, Ck 14, Ck 19 and vimentin staining according to the manufacturer's instructions. All antibodies were detected by a standard avidin-biotin complex method with a biotinylated rabbit anti-mouse antibody (DAKO) and an avidin-biotin complex (DAKO). The stainings were developed with diaminobenzidine or alternatively using LSAB/AP (DAKO). Before the slides were mounted, all sections were counterstained for 45 sec- onds with hematoxylin and dehydrated in alcohol and xylene. Appropriate negative controls (obtained by omis- Clone D5/16B4 K8.8/DC10 34βB4 DE-K10 LL002 KS19.1 V9 Pre-treatment Autoclave 10 min Autoclave 10 min, citrate buffer Steamer 30 min, citrate buffer Steamer 30 min, citrate buffer Steamer 30 min, citrate buffer Steamer 30 min, citrate buffer Steamer 30 min, citrate buffer Dilution 1 : 80 1 : 40 1 : 150 1 : 400 1 : 50 1 : 80 1 : 1000