Use chlorofom-methanol-water mixture...Chloroform phase will hold the most of the paraffin and many embedded lipophilic substances. Mid phase will present the protein composition and upper phase methanolic mixture contains most of the polar and mid polar molecules...If you do not consider lipidomic research, it would be better to move on with upper phase only...You may pretreat FFPE to remove most of the wax prior to metabolomic extraction and do not forget some of the molecules mainly lipophilic ones will be washed out through this removal process..

Procedure: (I work in natural product metabolomics but not in biology. Therefore, it's crucial to be mindful of sample denaturation and proceed with necessary precautions.)

1. Slide Deparaffinization:

Deparaffinize FFPE slices by incubating them in xylene. Repeat this step until the paraffin is completely removed.

2. Rehydration:

Gradually rehydrate the tissue slices through a series of ethanol washes (e.g., 100%, 95%, 70% ethanol).

3. Proteinase K Digestion:

Prepare a proteinase K digestion buffer and incubate the rehydrated tissue slices in this buffer containing proteinase K.

Incubate the samples overnight at a suitable temperature to allow digestion of proteins and reversal of cross-links.

4. Nucleic Acid Extraction:

Extract nucleic acids using a phenol:chloroform:isoamyl alcohol solution. Perform centrifugation to separate the aqueous phase containing nucleic acids.

Transfer the aqueous phase to a new tube and repeat the extraction with chloroform.

Precipitate nucleic acids by adding isopropanol and washing with ethanol.

5. Washing and Resuspension:

Wash the nucleic acid pellet with 70% ethanol to remove impurities.

Air-dry or speed-vac the pellet and resuspend it in RNase-free water.

Can I preserve the FFPE tissue in Ethanol after dewaxing and then extract the metabolites the next day?