Evaluation of ethanol‐fixed, paraffin‐embedded tissues for proteomic applications

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.

Comparison of Snap Freezing versus Ethanol Fixation for Gene Expression Profiling of Tissue Specimens

“…We have been able to generate proteomic data using microdissected cells from ethanol-paraffin samples. 3,16 How- ever, we have not assessed the global integrity of mRNA using a high-throughput approach such as microarray analysis.…”

Long-term preservation of antigenicity on tissue microarrays

“…These include HOPE fixative, 9 zinc fixation. 10,11 ethanol, 12,13 and UMFIX. 14 These fixatives show great promise in overcoming the deleterious effects of formalin-based fixation, and may also improve the durability of precut slides.…”

Protein unlocking procedures of formalin‐fixed paraffin‐embedded tissues: Application to MALDI‐TOF Imaging MS investigations

“…Tissue fixation through the formation of protein crosslinks (resulting from the formaldehyde reactivity with side-chain residues, e.g., lysine, asparagines, and glutamine) stabilize the cellular morphology preventing autolytic phenomena and organelles redistribution inside the cells [1,2]. Nevertheless as a major drawback it renders impossible the application of the routinely used procedures of biochemical protein extraction and the subsequent protein structural analysis [3,4].…”

“…In the field of proteomics investigations Ahram et al. [3] demonstrated that the proteins can be extracted from ethanol-fixed and paraffin-embedded tissue, and resolved by 2-DE separation. Recently Shi et al [4] obtained a satisfying overlapping between proteins identified from FFPE and frozen tissues and encouraging results are reported also by Prieto et al [5] and Crockett et al [6] that identified proteins from FFPE enzymatically digested tissues.…”