Ethanol-fixed cell/tissue samples

The sample materials from which proteins for proteomics studies may be extracted include fresh or snap-frozen cells from varied sources such as biological fluids, (serum, urine, plasma) and solid tissues such as biopsy specimens. Moreover, proteins isolated from ethanol-fixed paraffin-embedded tissues can be utilized for MS analysis.2 Protocols for the identification of proteins from formalin-fixed paraffin-embedded (FFPE) tissues have been recently developed.3 4 FFPE materials are the most common forms of biopsy archives utilized worldwide, and represent an important advancement for the large-scale interrogation of proteins in archival patient-derived materials. Finally, laser capture microdissected tissues have been successfully used for MS analysis.45... 

Cell culture, human or animal cellular sample. Satisfactory tissue samples for protein analysis are (1) frozen tissue sections, (2) ethanol-fixed, paraffin-embedded tissue, or laser capture microdissected (LCM) cell populations from the aforementioned tissues (see Note 1). 

Two-dimensional capillary electrophoresis of complex protein samples requires careful attention to detail. Tissues and cells should be fixed to prevent degradation. Most conventional fixatives are inappropriate because they produce covalent crosslinks, which are difficult to reverse. We find that ethanol produces decent results when the sample is homogenized with high concentrations of SDS. 

As a first step toward optimizing Immuno-LCM with alcohol fixation, we compared four different alcohol treatments. Serial cryosections of rat hindbrain were fixed in ice-cold 100% methanol, 70% methanol, 100% ethanol, or 70% ethanol for 2 min. The slides were treated for 1 min in cold PBS followed by dehydration as described above. A comparison of the amount of tissue removed by the AutoPix after these different conditions suggests that the different fixations resulted in visibly different amounts of tissue removed from the sections. All, however, produce samples that are suitable for efficient cell picking (Fig. 3). It appears, though, that fixation in 70% alcohol produced better tissue recovery in this experiment. 

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