Application of ImmunoMemBlot Method for the Analysis of Denatured Protein Extracts from Paraffin Sections
Sang Shin Lee1, Young Joon Lee1, Jae Geel Lim1, Ji Young Song1, Cheol Soo Yun1, Seong Hee Ko2, Yeon Sook Kim1, Suk Keun Lee1*
1Department of Oral Pathology, 2Department of Pharmacology, College of Dentistry, Kangnung National University, and Research Institute of Oral Science
Although many different methods for the genetic analyses of pathological lesions were developed, the formalin fixed tissues including paraffin sections were usually employed in the histological and immunohistochemical detections but almost limited in the quantitative analysis. However, for example, the protein in the paraffin section also contains same amount of epitopes as the native tissue product. In this study an ImmunoMemBlot (IMB) method was applied to detect the epitope of paraffin section for the comparative or quantitative analysis. Different from ordinary dot/slot blot methods, the IMB was designed to process the membrane blotting and immuno-visualization procedure directly in the wells of the apparatus, so that the time for the whole procedures can be shortened into less than 2 hours. Here, we performed the IMB for the analysis of comparative gene profile between ameloblastoma and adenomatoid odontogenic tumor. Simply, 10 microsections in 100 μm thickness were used for protein extract with protein lysis buffer of paraffin tissue, and followed by the IMB procedure. Ameloblastoma showed increased expressions of PCNA, CDK4, C-erbB2, CEA, p53, FLIP, FAS, Bcl-2, p21, EGFR, HGF, VEGF, N-Ras, MMP-2, MMP-9, Caspase-3, Caspase-8, and Caspase-9 than adenomatoid odontogenic tumor, while the latter showed more expressions of Bax, Bad, FADD, p21, E-cadherin, and TGF-β1 than the former. Therefore, it is considerable that ameloblastoma grows more rapidly with more frequent apoptotic tissue degeneration than adenomatoid odontogenic tumor, which highly expresses the genes of antitumor and cytodifferentiation. The present study indicates that IMB analysis can be applied for the primary screening of comparative gene profile using paraffin sections routinely prepared in pathology laboratory.