After antigen retrieval using 10?mM citrate buffer, pH?6

After antigen retrieval using 10?mM citrate buffer, pH?6.0, in a domestic pressure cooker (model Eterna 4??L; Nigro, Araraquara, Brazil) for 4?min, endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 20?min. Conclusions These results suggest that ezrin and podoplanin may contribute to the expansive growth and local invasiveness of keratocystic odontogenic tumors. Additionally, as both proteins were overexpressed by odontogenic epithelium, their possible roles need to be further explored in benign odontogenic tumors. strong class=”kwd-title” Keywords: Keratocystic odontogenic tumor, Ezrin, Podoplanin Background Podoplanin expression has been detected in epithelial cells of developing tooth germ [1, 2] and in odontogenic epithelium of benign tumors [3C10]. The presence of podoplanin in human tooth germ tissues, adult teeth and cystic odontogenic lesions suggested that this protein probably is involved in mechanisms of cell adhesion, epithelial-mesenchymal transition and invasion, and expansive growth of cystic odontogenic lesions [5]. Previous studies conducted in our laboratory investigated the association of podoplanin with cellular proliferative activity, determined by Ki-67 antibody, in ameloblastomas [7] and keratocystic odontogenic tumors [9]. We did not find a statistically significant correlation in ameloblastomas, however this association was observed in keratocystic odontogenic tumors. Moreover, both podoplanin and Ki-67 expressions were stronger and co-localized in keratocystic odontogenic tumors when compared to the orthokeratinized odontogenic cysts, an indolent lesion. These fingings suggested that podoplanin positive cells are located in the cell proliferation centre indicating a role for this protein in the process of tumoral invasion [9]. Furthermore, Friedrich et al. [8] showed that podoplanin expression pattern is similar between keratocyst odontogenic tumor diagnosed in sporadic and in nevoid basal cell carcinoma syndrome and, the authors reinforced the probable association of this protein with invasion and Lomerizine dihydrochloride local recurrences of the tumor. Recent findings prove that podoplanin is important to drive directional cell migration in epithelial and tumor Lomerizine dihydrochloride cells [11]. Then, the ability of podoplanin to remodel cytoskeleton and form filipodia-like membrane extension [12] has been suggested as important factor in movement of odontogenic epithelial cells [6]. This podoplanin-induced cell motility through of actin cytoskeleton rearrangement seems to be dependent on the interaction with the cytoplasmatic Lomerizine dihydrochloride ezrin [13], a member of ERM (ezrin, radixin, moesin) protein family protein [14, 15]. The currently study was designed to analyze the immunolocalization of ezrin and its relationship with Lomerizine dihydrochloride podoplanin expression in keratocystic odontogenic tumors. To the best of our knowledge, this is the first report of ezrin immunostaining in an odontogenic tumor. Methods Patients and tumor samples All surgical specimens of keratocystic odontogenic tumor analyzed in this study were obtained from the Laboratory of Pathology, Bauru Rabbit Polyclonal to Claudin 3 (phospho-Tyr219) School of Dentistry, University of S?o Paulo, between 2002 and 2010. The inclusion criteria were: i) patients with diagnosis of keratocystic odontogenic tumor based on the classification of the World Health Organization [16], determined by the sum of the clinical, radiographic, and microscopic data; ii) availability of the paraffin block with Lomerizine dihydrochloride sufficient and representative amount of odontogenic tumor for microscopic analysis. Applying the inclusion criteria, 18 keratocystic odontogenic tumors were selected for investigation of podoplanin and ezrin immunostaining. This study was approved by the Research Ethics Committee of the Bauru School of Dentistry, University of S?o Paulo (process #85612/2012). Immunohistochemistry Formalin-fixed 3?m sections of keratocystic odontogenic tumors were obtained from the pathology archive for immunohistochemistry analysis of the ezrin and podoplanin expressions by odontogenic epithelium. After antigen retrieval using 10?mM citrate buffer, pH?6.0, in a domestic pressure cooker (model Eterna 4??L; Nigro, Araraquara, Brazil) for 4?min, endogenous peroxidase activity was blocked by incubation in 3% H2O2 for 20?min. Each section was incubated overnight at 48C with the primary monoclonal anti-podoplanin antibody (D2-40 clone, code#3619-1; Dako North America, Inc., Carpinteria, CA, USA), dilution 1:200 or anti-ezrin antibody (Dako North America, Inc., CA, USA), dilution 1:1000,.