Evaluation of ethanol-fixed, paraffin-embedded tissues for proteomic applications.
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.
Ahram
Mamoun
M
Pathogenetics Unit, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Flaig
Michael J
MJ
Gillespie
John W
JW
Duray
Paul H
PH
Linehan
W Marston
WM
Ornstein
David K
DK
Niu
Shulan
S
Zhao
Yingming
Y
Petricoin
Emanuel F
EF
Emmert-Buck
Michael R
MR
D015180:Electrophoresis, Gel, Two-Dimensional; D004801:Eosine Yellowish-(YS); D000431:Ethanol; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D013058:Mass Spectrometry; D042282:Microdissection; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D016612:Paraffin Embedding / Q000379:methods*; D011471:Prostatic Neoplasms / Q000737:chemistry / Q000473:pathology; D040901:Proteomics / Q000379:methods*; D013194:Staining and Labeling; D016707:Tissue Fixation / Q000379:methods*; D014992:Xylenes
D009363:Neoplasm Proteins; D014992:Xylenes; D000431:Ethanol; D004801:Eosine Yellowish-(YS)