TEM: B-glucuronidase localization

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From:"Jaci Lett" <jmlett@cid.wustl.edu>
To:"Microscopy Listserver" <Microscopy@sparc5.microscopy.com>, "Histonet Listserver" <Histonet@pathology.swmed.edu>
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We are looking to localize B-glucuronidase in the mouse cochlea.  We'd like
to stain for the enzyme and still be able to decalcify the cochleas and
process them into Epon-Araldite for LM and TEM without losing the reaction
product.  Does anyone have any suggestions for staining or localization
protocols for this enzyme?  I'm not certain whether the tissue will be taken
all the way to TEM, but I do know we'll be looking at LM (1 to 4 microns).
I found a method by Hayashi and Hayashi ("Electron Microscopic Cytochemistry
and Immunocytochemistry in Biomedicine", CRC Press, 1993) using hexazonium
pararosanilin (red in LM and electron dense for EM) on 40 micron non-frozen
sections (and then processed for EM) that sounds appropriate, but I'm
concerned that it may not work en bloc (the cochlea is a twisty, windy

I posted this request a few months ago and received no assistance (just
individuals also interested in similar protocols and a company promoting
their antibody).

Thanks so much for any direction I can get,

Jaclynn M. Lett, Research Assistant     jmlett@cid.wustl.edu

Fay and Carl Simons Center for Biology of Hearing and Deafness
Central Institute for the Deaf
818 S. Euclid Ave.
St. Louis, MO 63110

voice:  314-977-0257     fax:  314-977-0030

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