[Histonet] Wax removal incomplete

From:John Kiernan

A recent publication from Ireland shows (by laser confocal 
Raman microspectroscopy, no less) that wax removal from 
sections, as usually practised, is always incomplete. 
The authors were able to remove all the paraffin, however, 
with hexane. The other things they tried were xylene, 
"histoclear" followed by hot antigen retrieval, and 
"trilogy" (a product that combines dewaxing and retrieval 
- ? a solvent-detergent mixture). All three failed to 
remove all the wax, even with repeated, prolonged
For hexane, 18 hours were needed for complete was removal. 

The investigators found that the residual paraffin
seriously interfered with the Raman spectra of  
rehydrated sections. They are investigating the Raman
technique (which they explain fairly simply) for early 
diagnosis of tumours. They also claim stronger
immunostaining after dewaxing in hexane (18h) than after 
xylene (18h). They tested only one antibody (to
and the pair of photos doesn't make the point very

They authors also state that hexane is less toxic than 
xylene. I can add that the least expensive n-hexane is 
a little cheaper than xylenes (mixed isomers) in one 
catalogue checked.  Both solvents are flammable, and
n-hexane (BP 69C) is more volatile than xylene. Both
solvents are miscible with 100% alcohol.

The reference is:
O'Faolain E et 6 al. (2005) Raman spectroscopic evaluation
of efficacy of current paraffin wax section dewaxing
agents. J. Histochem. Cytochem. 53(1): 121-129.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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