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Evaluation of ethanol-fixed,paraffin-embedded tissues for proteomic applications
Authors:Ahram Mamoun  Flaig Michael J  Gillespie John W  Duray Paul H  Linehan W Marston  Ornstein David K  Niu Shulan  Zhao Yingming  Petricoin Emanuel F  Emmert-Buck Michael R
Institution:Pathogenetics Unit, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Abstract:We previously reported that ethanol fixation and paraffin embedding of tissues produce excellent histomorphology and good preservation of macromolecules. Here, we present a detailed evaluation of ethanol-fixed tissues for proteomic initiatives. When proteins were extracted from ethanol-fixed, paraffin-embedded prostate tissue, resolved by two-dimensional gel electrophoresis (2-DE), and stained by standard methods, several hundred protein molecules could be detected and successfully analyzed by mass spectrometry. Protein profiles obtained from ethanol-fixed tissues were highly similar to those observed from frozen tissues, in contrast to the poor protein recovery from formalin-fixed material. The protein content of specific cells that were microdissected from ethanol-fixed tissue sections using laser capture microdissection could also be successfully analyzed by 2-DE. We observed that eosin staining of tissue sections had a detrimental effect on protein separation, whereas hematoxylin staining had minimal consequence. In order to illustrate the applicability of ethanol-fixed tissues for proteomic discovery studies, we compared the protein profiles of patient-matched, normal prostatic epithelial cells and invasive adenocarcinoma cells obtained from ethanol-fixed, paraffin-embedded tissues. A number of differentially expressed proteins was discovered and identified by mass spectrometry. Immunohistochemical analyses performed on ethanol-fixed tissue sections were in agreement with the proteomic discovery findings. In light of these results, we conclude that ethanol-fixed tissues can be successfully utilized for proteomic analyses.
Keywords:Ethanol fixation  Laser capture microdissection  Prostate cancer  Tissue staining
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