I could just be displaying my ignorance here, but if the problem is the
prescence of polymerised plastics in the wax, then a technique sometimes
used for EM might help. To remove polymerised epoxy resins from a block or
from thick sections, one exposes them to sodium ethoxide or sodium methoxide
for a short time. Either is made very easily by adding sodium hydroxide
pellets to absolute ethanol or methanol and leaving it overnight in the fume
hood. Since it would be applied to the ex-paraffin sections, you wouldn't
have to leave them in for very long, so there shouldn't be too much damage
to the tissue, though I've never tried immuno-staining after using this. It
would probably work best (if it works at all) after removing the wax with
xylene or whatever and rinsing well in 100% alcohol.

Lesley Weston.



on 15/02/2005 6:08 AM, LEWIS, MARK A. at mark.lewis@thermo.com wrote:

> I often wondered about this due to the "plastic "polymers added to the
> different types of paraffins. Perhaps it's these "polymers" that are not being
> removed with the typical solvents.
> 
> Interesting ...
> 
> Thanks John !
> 
> Mark
>  
> Mark A. Lewis B.A., H.T.(ASCP)
> Product Specialist
> Anatomical Pathology
> Clinical Diagnostics
> Thermo Electron Corporation
> 171 Industry Drive
> Pittsburgh, Pa. 15275
> Phone:  412-747-4013
> Fax       412-788-6557
> E-mail: mark.lewis@thermo.com
> 
> 
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu
> [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of John Kiernan
> Sent: Monday, February 14, 2005 1:40 PM
> To: Histonet
> Subject: [Histonet] Wax removal incomplete
> 
> 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
> treatments. 
> 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
> cytokeratin), 
> and the pair of photos doesn't make the point very
> impressively. 
> 
> 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.


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